Gene-targeted non-human mammal with human FAD presenilin mutation and generational offspring

ABSTRACT

The present invention provides a gene-targeted, non-human mammal having a gene encoding a mutant protein product of a mutated FAD presenilin-1 (PS-1) gene, a human FAD Swedish mutation, and a humanized Aβ mutation, and generational offspring thereof and a gene-targeted, non-human mammal having a gene encoding a mutant protein product of a mutated FAD PS-1 gene and a human Swedish APP695 mutation, and generational offspring thereof, as well as methods of identifying compounds useful in treating Alzheimer&#39;s disease, and to methods of treating Alzheimer&#39;s disease.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part and claimspriority under 35 U.S.C. § 120 to U.S. Ser. No. 09/041,185 filed Mar.10, 1998, which claims priority under 35 U.S.C. § 119(e) to ProvisionalSerial No. 60/057,069 filed Aug. 29, 1997, each of which areincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to gene-targeted, non-human mammalscomprising a human mutation in the non-human mammalian presenilin 1(PS-1) FAD gene, methods of identifying compounds for treatingAlzheimer's disease, and to methods of treating Alzheimer's disease.

BACKGROUND OF THE INVENTION

[0003] Alzheimer's disease (AD) is an age-dependent neurodegenerativedisorder that leads to profound behavioral changes and dementia.Hallmark pathologies include the atrophy of brain gray matter as aresult of the massive loss of neurons and synapses, and proteindeposition in the form of both intraneuronal neurofibrillary tangles andextracellular amyloid plaques within the brain parenchyma. In addition,affected areas of the AD brain exhibit a reactive gliosis that appearsto be a response to brain injury. Surviving neurons from vulnerablepopulations in AD show signs of metabolic compromise as indicated byalterations in the cytoskeleton (Wang et al., Nature Med., 1996, 2,871-875), Golgi complex (Salehi et al., J. Neuropath. Exp. Neurol.,1995, 54, 704-709) and the endosomal-lysosomal system (Cataldo et al.,Neuron, 1995, 14, 671-680).

[0004] Approximately 10 to 30% of AD cases are inherited in an autosomaldominant fashion and are referred to as “familial Alzheimer's disease”or “FAD.” Genetic linkage studies have revealed that FAD isheterogeneous and a majority of the cases have been linked to genemutations on chromosomes 1, 14, 19, or 21 (reviewed in Siman and Scott,Curr. Opin. Biotech., 1996, 7, 601-607). Importantly, these individualshave been shown to develop the classical symptomatic and pathologicalprofiles of the disease confirming that the mutations are associatedwith the development of the disease rather than a related syndrome. Thelocus on chromosome 14 is associated with a significant fraction of FAD,and mutations at the locus have been mapped to a single-copy gene,termed “S182” or “presenilin 1” (PS-1), that encodes a 467 amino acidprotein (Sherrington et al., Nature, 1995, 375, 754-760; Clark et al.,Nature Genet., 1995, 11, 219-222). A closely related gene, “STM2” or“presenilin 2” (PS-2), located on chromosome 1, has been linked to twoadditional FAD kindreds including the descendants of German familiesfrom the Volga valley of Russia (Levy-Lahad et al., Science, 1995, 269,973-977; Rogaev et al., Nature, 1995, 376, 775-778). PS-1 and PS-2 sharean overall 67% amino acid sequence homology, and primary structureanalysis indicates they are integral membrane proteins with 6 to 8trans-membrane domains (Slunt et al., Amyloid-Int. J Exp. Clin. Invest.,1995, 2, 188-190; Doan et al., Neuron, 1996, 17, 1023-1030). Much of theinformation on function of the presenilins stems from the identificationof a presenilin homolog in C. elegans termed “SEL-12,” a 6 to 8trans-membrane protein that appears to participate in an intracellularsignaling pathway mediated by the lin-12/glp-1/Notch family (Levitan andGreenwald, Nature, 1995, 377, 351-354). PS-1 and SEL-12 proteins share a49% sequence homology and have similar membrane orientations.Importantly, both human PS-1 and PS-2 can rescue the mutant sel-12phenotype in C. elegans, indicating a role for the presenilins in Notchsignaling (Levitan et al., Proc. Natl. Acad. Sci. USA, 1996, 93,14940-14944).

[0005] FAD linked to the presenilins is highly penetrant and theaggressive nature of the disease suggests that the mutant proteinparticipates in a seminal pathway of AD pathology. To date, over seventyFAD mutations have been identified in PS-1, and three FAD mutations havebeen found in PS-2. Most of the FAD mutations occur in conservedpositions between the two presenilin proteins, suggesting that they areaffecting functionally or structurally important amino acid residues.Interestingly, many of the mutated amino acids are also conserved inSEL-12. All but two of the presenilin mutations are missense mutations.One exception results in an aberrant RNA splicing event that eliminatesexon 9, creating an internally-deleted mutant protein (Perez-Tur et al.,NeuroReport, 1995, 7, 297-301; Sato et al., Hum. Mutat. Suppl., 1998, 1,S91-94; and Prihar et al., Nature Med., 1999, 5, 1090). The otherresults in two deletion transcripts (Δ4 and Δ4cryptic) and onefull-length transcript with the amino acid Thr inserted between 113 and114 (DeJonghe et al., Hum. Molec. Genet., 1999, 8, 1529-1540). Thelatter transcript leads to the AD pathophysiology. These latter points,along with the genetic dominance of the disease, argue that diseasepathogenesis in the presenilin kindreds requires the production of amutant presenilin protein having a novel and detrimental function,rather than the simple loss or reduction of normal presenilin levels.The mutations do appear to disrupt normal presenilin function however,because mutant presenilins are not able to rescue or fully rescue thesel-12 phenotype (Levitan et al., Proc. Natl. Acad. Sci. USA, 1996, 93,14940-14944).

[0006] Expression profiles of the presenilins have been examined at agross level but, so far, these analyses have yielded little informationon the mechanism of disease pathogenesis. Both presenilin 1 and 2 arewidely expressed in the CNS and peripheral tissues. In brain, expressionis enriched in neurons but is apparent in both AD-vulnerable andresistant areas. Cellular localization studies indicate that theproteins accumulate primarily in the Golgi complex and endoplasmicreticulum but no significant alterations in expression levels orsubcellular distribution have been attributed to the FAD mutations(Kovacs et al., Nature Med., 1996, 2, 224-229).

[0007] The presenilin proteins are processed proteolytically through twointracellular pathways. Under normal conditions, accumulation of 30 kDN-terminal and 20 kD C-terminal proteolytic fragments occurs in theabsence of the full-length protein. This processing pathway is highlyregulated and appears to be relatively slow, accounting for turnover ofonly a minor fraction of the full-length protein. The remaining fractionappears to be rapidly degraded in a second pathway by the proteasome(Thinakaran et al., Neuron, 1996, 17, 181-190; Kim et al., J. Biol.Chem., 1997, 272, 11006-11010). Proteolytic metabolism of PS-1 variantslinked to FAD appears to be different, but the relevance of the changeto pathogenesis is not known (Lee, et al., Nature Med., 1997, 3,756-760).

[0008] One pathogenic role for the mutant presenilins in FAD appears tobe related to effects on processing of the amyloid precursor protein(APP) and production of the Aβ peptide, the primary proteinaceouscomponent of the extaacellular neuritic plaque in the AD brain. Elevatedserum levels of the longer form of Aβ (Aβ42), considered to be the morepathogenic species of the Aβ peptides, have been measured in patientsbearing PS-1 and PS-2 mutations (Scheuner et al., Nature Med., 1996, 2,864-870). Additionally, FAD brains with PS-1 mutations have largeamounts of Aβ deposition (Lemere et al., Nature Med., 1996, 2,1146-1150; Mann et al., Ann. Neurol., 1996, 40, 149-156; Gómez-Isla etal., Ann. Neurol., 1997, 41, 809-813). Elevated levels of Aβ1-42 werealso found in cells transfected with mutant PS-1 or PS-2 and in miceexpressing mutant PS-1 (Borchelt et al., Neuron, 1996, 17, 1005-1013;Duff et al., Nature, 1996, 383, 710-713; Citron et al., Nature Med.,1997, 3, 67-72; Murayama et al., Prog. Neuro-Psychopharmacol. Biol.Psychiatr., 1999, 23, 905-913; Murayama et al., Neurosci. Lett., 1999,265, 61-63; Nakano et al., Eur. J. Neurosci., 1999, 11, 2577-2581). Themechanism by which the mutant presenilins affect APP processing is notknown, but these results do support a causative role of increased Aβ42production in the development of FAD. Importantly, it is possible thatmutant presenilins influence other AD pathogenic processes as well, suchas presumptive intracellular signaling and cell death pathways involveddirectly or indirectly in neuronal dysfunction and degeneration.

[0009] Genetically-engineered animals have been used extensively toexamine the function of specific gene products in vivo and their role inthe development of disease phenotypes. The genetic engineering of micecan be accomplished according to at least two distinct approaches: (1) atransgenic approach where an exogenous gene is randomly inserted intothe host genome, and (2) a gene-targeting approach where a specificendogenous DNA sequence or gene is partially or completely removed, orreplaced by homologous recombination. The transgene of a transgenicorganism is comprised generally of a DNA sequence encoding the proteinsequence and a promoter that directs expression of the protein codingsequences. A transgenic organism expresses the transgene in addition toall normally-expressed native genes. The targeted gene of agene-targeted animal, on the other hand, can be modified in one of twoways: (1) a functional form where a modified version of the targetedgene is expressed, or (2) a non-functional or “null” form where thetargeted gene has been disrupted resulting in loss or reduction ofexpression. If the targeted gene is a single copy gene and the animal ishomozygous at the targeted locus, then, depending on the type ofmodification, the animal either does not express the targeted gene orexpresses only a modified version of the targeted gene in absence of thenormal form.

[0010] Transgenic mice expressing native and mutant forms of thepresenilin proteins have been described (Borchelt et al., Neuron, 1996,17, 1005-1013; Duff et al., Nature, 1996, 383, 710-713; Borchelt et al.,Neuron, 1997, 19, 939-945; Citron et al., Nature Med., 1997, 3, 67-72;Chui et al., Nature Med., 1999, 5, 560-564; and Nakano et al., Eur. J.Neurosci., 1999, 11, 2577-2581). Although mice bearing mutations in PS-1had elevated levels of Aβ1-42, they have not formed Aβ depositscharacteristic of AD or shown behavioral deficits associated with AD.Neuronal loss has been described by one group (Chui et al., Nature Med.,1999, 5, 560-564). When transgenic mice with PS-1 mutations were crossedwith transgenic mice bearing the Swedish APP mutations, there was markedacceleration in the formation of Aβ deposits (Borchelt et al., Neuron,1997, 19, 939-945; Holcomb et al., Nature Med., 1998, 4, 97-100; Lamb etal., Nature Neurosci., 1999, 2, 695-697). Gene-targeted PS-1 null micelacking one or both functional alleles of the PS-1 gene have also beendescribed (Wong et al., Nature, 1997, 387, 288-292, and Shen et al.,Cell, 1997, 89, 629-639). Mice in which both PS-1 alleles have beendisrupted resulting in the complete loss of PS-1 expression are notviable and die shortly after birth. No abnormal phenotypes or changes inAPP processing have been reported in mice lacking only one of the twoPS-1 alleles, but inhibition of APP processing is found in neuronsderived from PS-1 null mice (DeStrooper et al., Nature, 1998, 391,387-390).

[0011] In the present application, a gene-targeting approach (Reaume etal., J. Biol. Chem., 1996, 271, 23380-23388, which is incorporatedherein by reference in its entirety) generating AD models is described.One model employs the Swedish FAD mutation and “humanized” mouse Aβsequence in the APP gene (U.S. Pat. No. 5,777,194, which is incorporatedherein by reference in its entirety). This mouse (APP^(NLh/NLh))produced normal levels of APP, overproduced human Aβ1-40 and 1-42, butdid not deposit Aβ (Reaume et al., J. Biol. Chem., 1996, 271,23380-23388). A human PS-1 mutation, the P264L mutation in particular,was introduced into the mouse PS-1 gene. The P264L mutation is anon-conservative amino acid substitution in the cluster of mutations inexon 8, causing an onset of FAD in the middle forties to middle fifties(Campion et al., Hum. Molec. Genet., 1995, 4, 2373-2377; Wasco et al.,Nature Med., 1995, 1, 848). Crosses producedAPP^(NLh/NLh)×PS-1^(P264L/P264L) double gene-targeted mice. These micehad elevated levels of Aβ1-42, sufficient to cause Aβ deposition. Micebearing the PS-1^(P264L) mutation were also crossed with Tg2576 micethat overexpress Swedish APP695 (Hsiao et al., Science, 1996, 274,99-102; available from the Mayo Clinic, Rochester, Minn.). One distinctadvantage of the present invention is that for heterozygous andhomozygous gene-targeted mice, the fidelity of expression patterns ofproteins is maintained since the expression is under the endogenouspromoter. Further, expression levels of the holoprotein are not changed.

SUMMARY OF THE INVENTION

[0012] The present invention relates to a gene-targeted, non-humanmammal comprising a gene encoding a mutant protein product of a mutatedFAD presenilin-1 (PS-1) gene, a human FAD Swedish mutation, and ahumanized Aβ mutation, and generational offspring thereof. The presentinvention also relates to a gene-targeted, non-human mammal comprising agene encoding a mutant protein product of a mutated FAD presenilin-1(PS-1) gene and a human Swedish APP695 mutation, and generationaloffspring thereof. Preferably, the PS-1 gene has been mutated to containthe human P264L mutation (Wasco et al., Nature Medicine, 1995, 1, 848).In particular, the present invention relates to a mouse wherein a partof a mouse presenilin 1 gene encoding presenilin 1 protein has beenreplaced with a DNA sequence that results in a mouse presenilin 1 genethat contains a human mutation, most preferably a P264L mutation. Stillmore specifically, the base sequence of codon 264 of the mousepresenilin 1 gene is altered from CCG to CTT, which is the base sequencefound to constitute the P264L mutation of humans. The mutated gene codonencodes leucine in place of proline in amino acid number 264 ofpresenilin 1. Additionally, and still more specifically, a nucleotidebase in codon 265 of the mouse presenilin 1 gene is altered fromadenosine to guanosine, but this change does not result in an amino acidchange in the expressed protein. However, the combined sequence ofcodons 264 and 265, after the incorporation of the most preferredchanges described above, results in a restriction enzyme site for therestriction enzyme AflII.

[0013] Accordingly, in one embodiment, the present invention features anon-human mammal and generational offspring homozygous for a targetedmutant PS-1 gene comprising a mutated FAD gene preferably a mousepresenilin 1 protein-encoding sequence comprising a human mutation, mostpreferably a P264L mutation, in place of the native presenilin 1protein-encoding sequence. In another embodiment, the invention featuresa non-human mammal and generational offspring heterozygous for atargeted PS-1 gene comprising a mutated mouse FAD gene, preferably amouse presenilin 1 protein-encoding sequence containing a humanmutation, most preferably a P264L mutation, in place of the nativepresenilin 1 protein-encoding sequence.

[0014] The present invention is also directed to methods for identifyinga compound for treating Alzheimer's disease comprising administering acompound to a mammal heterozygous or homozygous for a mutation of thePS-1 gene and a human Swedish APP695 mutation, or generational offspringthereof, or to a mammal heterozygous or homozygous for a mutation of thePS-1 gene, a human FAD Swedish mutation, and a humanized Aβ mutation,and generational offspring thereof, and measuring the amount of Aβ42peptide in a tissue sample from the mammal.

[0015] The present invention is also directed to methods of treating anindividual suspected of having Alzheimer's disease comprisingadministering to the individual an effective Alzheimer's diseasetreatment amount of a compound identified by the method described above.

[0016] The present invention is also directed to compounds identified byany of the methods described above

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1 is a schematic diagram illustrating general principles ofgene targeting.

[0018]FIG. 2 is a set of mouse PS-1 genomic clone maps prepared usingthe Flash™ Non-radioactive Gene Mapping Kit. Letter abbreviations forrestriction endonucleases are as follows: E, EcoRl; X, Xbal; H, HindIII;B, BamHI; Xh, Xhol.

[0019]FIG. 3 is a representative restriction map used to illustrate aFlash™ restriction mapping method.

[0020]FIG. 4 is a diagram illustrating the strategy for placing exons 7and 8 on the restriction map of PS-1.

[0021]FIG. 5 is a pair of genetic maps illustrating the relationshipbetween Exon 8 of PS-1 and the pPS1-8-TV replacement vector. Letterabbreviations for restriction endonucleases are as follows: E, EcoRl; X,Xbal; H, HindIII; B, BamHl; Xh, Xhol, N, NotI.

[0022]FIG. 6 is a schematic diagram illustrating the construction ofplasmid pPNTIox².

[0023]FIG. 7 is a schematic diagram illustrating the construction ofplasmid pPS1-XH16.

[0024]FIG. 8 is a schematic diagram illustrating the construction ofplasmid pPS1-XB1.

[0025]FIG. 9 is a schematic diagram illustrating the construction ofplasmids pPS1-X15 and pPS1-X2.

[0026]FIG. 10 is a schematic diagram illustrating the construction ofplasmid pPS1-X319.

[0027]FIG. 11 is a schematic diagram illustrating the restrictionmapping of the 5′ Arm of Homology from plasmids pPS1-X15 and pPS1-X2.

[0028]FIG. 12 is a pair of restriction maps for the PS1 3′ and 5′ armsof homology.

[0029]FIG. 13 is a partial sequence of exon 8 of PS-1 illustrating thebase changes to effect the P264L mutation and the addition of the AflIIrestriction endonuclease site of this invention.

[0030]FIG. 14 is a schematic diagram illustrating the construction ofplasmid pPS1-XB85.

[0031]FIG. 15 is a schematic diagram illustrating the construction ofplasmid pPS1-206.

[0032]FIG. 16 is a schematic diagram illustrating the construction ofplasmid pPS1-360.

[0033]FIG. 17 is a schematic diagram illustrating the construction ofplasmid pPNT3′413.

[0034]FIG. 18 is a schematic diagram illustrating the construction ofplasmid pPS1-8-TV.

[0035]FIG. 19 is a schematic diagram illustrating the strategy to detecthomologous recombination within mouse PS1. Letter abbreviations forrestriction endonucleases are as follows: E, EcoRI; X, XbaI; N NotI; H,HindIII; B, BamHI; A, Apal; Af, AflII; Sc, Scal; K, KpnI; and Hc,HincII.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0036] The present invention relates to a gene-targeted, non-humanmammal (and generational offspring of such mammal) that contains in thenon-human mammal's endogenous (i.e., native) genome presenilin 1 genethat comprises a human mutation, most preferably a human P264L mutation.The non-human mammal can also comprise a human FAD Swedish mutation anda humanized Aβ mutation. A non-human mammal can also comprise, inaddition to the human PS-1 mutation, a human Swedish APP695 mutation.Most preferably, the gene-targeted, non-human mammal produces a mutatedpresenilin 1 protein instead of the presenilin 1 protein normallyproduced by the non-human mammal. Gene-targeted, non-human mammalshomozygous for a presenilin 1 gene containing a human mutation, such asthe human P264L mutation, produce the mutated presenilin 1 proteinexclusively. Gene-targeted, non-human mammals heterozygous for aresenilin 1 gene containing a human mutation, such as the human P264Lmutation, produce both the mutated presenilin 1 protein and the nativepresenilin 1 protein. Preferably, the gene-targeted, non-human mammal ofthis invention is a rodent, and more specifically a mouse.

[0037] Importantly, because the non-human mammal of this invention isgenerated by gene targeting, as opposed to transgenic techniques, themammal produces the mutated presenilin 1 protein exclusively by normalendogenous presenilin 1 protein expression mechanisms. Advantageously,and unlike expression resulting from trarsgenic approaches, thepresenilin 1 protein is expressed from genes having the normal copynumber, and under the control of the endogenous presenilin 1 geneexpression control mechanisms. As a result, the presenilin 1 protein inthe non-human animal of this invention is produced with the samedevelopmental timing, same tissue specificity, and same rates ofsynthesis normally associated with native presenilin 1 protein in thewild-type, non-human mammal.

[0038] The gene-targeted, non-human mammals of this invention may beused as tools or models to elucidate the role of PS-1 comprising a humanmutation, preferably the human P264L mutation, in the pathology andsymptomatology of AD. They may be used to elucidate the manner in whichthe human mutation, preferably the P264L mutation, increases theproduction of the amyloid protein Aβ42. As used herein, the term“increase” when used in the foregoing context, means that the levels ofAβ42 produced by the non-human mammals disclosed herein are elevatedrelative to wild-type controls.

[0039] The non-human mammals of this invention and generationaloffspring also may be used as assay systems to screen for in vivoinhibitors and for discovering and testing the efficacy and suitabilityof putative chemical compounds for their ability to inhibit theformation, presence and deposition of excessive amounts of Aβ peptide inthe brain tissues, other tissues and body fluids (e.g., blood; plasma,and cerebrospinal fluid), said method comprising the steps of: (a)administering said chemical compounds to a non-human mammal homozygousor heterozygous for a targeted mutant PS-1 gene comprising a humanmutation, preferably the human P264L mutation, comprising: a mouse PS-1peptide encoding sequence containing a human mutation, preferably theP264L mutation, in place of the native PS-1 peptide encoding sequenceand (b) measuring the amounts of Aβ peptide in brain tissues, othertissues and body fluids (or some combination thereof) of said non-humanmammal, at an appropriate interval of time after the administration ofsaid chemical compounds.

[0040] As used in this disclosure, the following terms and phrases havethe following indicated definitions.

[0041] As used herein, “Aβ peptide” means either Aβ40 or Aβ42 orfragments thereof.

[0042] As used herein, “arms of homology” means nucleotide DNA sequencesin a targeting vector: (1) which have sufficient length and homology toprovide for site-specific integration of part of the targeting vectorinto the target gene by homologous recombination; (2) in which, orbetween which are located one or more mutations to be introduced into atarget gene; and (3) which flank a positive selectable marker.

[0043] As used herein, “homologous recombination” means rearrangement ofDNA segments, at a sequence-specific site (or sites), within or betweenDNA molecules, through base-pairing mechanisms.

[0044] As used herein, “human mutation in the non-human mammalianpresenilin 1 (PS-1) FAD gene” means any mutation of the PS-1 gene in anon-human mammal that results in the non-human mammal having anucleotide or nucleotides that correspond to the human PS-1 gene at thecorresponding position of the nucleotide or nucleotides. A humanmutation in the non-human mammalian presenilin 1 (PS-1) FAD geneincludes, but is not limited to, the following: A79V, V82L, V96F, Y115C,E120D, E120K, M139I, M139T, M139V, I143F, I143T, M146I, M146L (A

T), H163Y, G209V, A231T, A231V, M233T, L235P, L250S, A260V, L262F,C263R, P264L, P267S, R269H, R278T, E280A, E280G, A285V, E318G, G378E,G384A, and L392V, each of which is disclosed in Cruts et al., HumanMutat., 1998, 11, 183-190, which is incorporated herein by reference inits entirety; M146L (A

C) which is disclosed in Cruts et al., Human Mutat., 1998, 11, 183-190,Duff et al., Nature, 1996, 383, 710-713, Citron et al., Nature Med.,1997, 3, 67-72, Lee et al., Nature Med., 1997, 3, 756-760, and Lamb etal., Nature Neurosci., 1999, 2, 695-697, each of which is incorporatedherein by reference in its entirety; M146V, which is disclosed in Crutset al., Human Mutat., 1998, 11, 183-190, Duff et al., Nature, 1996, 383,710-713, and Guo et al., Nature Med., 1999, 5, 101-106, each of which isincorporated herein by reference in its entirety; H163R, which isdisclosed in Cruts et al., Human Mutat., 1998, 11, 183-190, Lamb et al.,Nature Neurosci., 1999, 2, 695-697, and Chui et al., Nature Med., 1999,5, 560-564, each of which is incorporated herein by reference in itsentirety; I213T, which is disclosed in Cruts et al., Human Mutat., 1998,11, 183-190 and Nakano et al., Eur. J. Neurosci., 1999, 11, 2577-2581,each of which is incorporated herein by reference in its entirety;L286V, which is disclosed in Cruts et al., Human Mutat., 1998, 11,183-190, Citron et al., Nature Med., 1997, 3, 67-72, and Chui et al.,Nature Med., 1999, 5, 560-564, each of which is incorporated herein byreference in its entirety; A246E, which is disclosed in Cruts et al.,Human Mutat., 1998, 11, 183-190, Lee et al., Nature Med., 1997, 3,756-760, and Qian et al., Neuron, 1998, 20, 611-617, each of which isincorporated herein by reference in its entirety; Y115H, which isdisclosed in Citron et al., Nature Med., 1997, 3, 67-72, which isincorporated herein by reference in its entirety; T116N, which isdisclosed in Romero et al., Neuroreport., 1999, 10, 2255-2260, which isincorporated herein by reference in its entirety; P117L and L171P, bothof which are disclosed in St. George Hyslop, Biol. Psychiatr., 2000, 47,183-199, which is incorporated herein by reference in its entirety;E123L, which is disclosed in Yasuda et al., Arch. Neurol., 1999, 56,65-69, which is incorporated herein by reference in its entirety; N135D,C410Y, A426P and P436S, each of which is disclosed in Hardy et al.,Trends Neurosci., 1997, 20, 154-159, which is incorporated herein byreference in its entirety; M139K, which is disclosed in Dumanchin etal., J. Med. Genet., 1998, 35, 672-673, which is incorporated herein byreference in its entirety; T147I, W165C, L173W, and S390I, each of whichis disclosed in Campion et al., Am. J. Human Genet., 1999, 65, 664-670,which is incorporated herein by reference in its entirety; L166R, whichis disclosed in Ezquerra et al., Arch. Neurol., 2000, 57, 485-488, whichis incorporated herein by reference in its entirety; S169L and P436Q,each of which is disclosed in Taddei et al., Neuroreport., 1998, 9,3335-3339, which is incorporated herein by reference in its entirety;S169P, which is disclosed in Ezquerra et al., Neurol., 1999, 52,566-570, which is incorporated herein by reference in its entirety;E184D, which is disclosed in Yasuda et al., Neurosci. Lett., 1997, 232,29-32, which is incorporated herein by reference in its entirety; G209R,which is disclosed in Sugiyarna et al., Online Human Mutat., 1999, 14,90, which is incorporated herein by reference in its entirety; L219P,which is disclosed in Smith et al., Neuroreport., 1999, 10, 503-507,which is incorporated herein by reference in its entirety; M233L andA409T, both of which are disclosed in Aldudo et al., Human Mutat., 1999,14, 433-439, which is incorporated herein by reference in its entirety;E273A, which is disclosed in Kamimura et al., J. Neurol. Sci., 1998,160, 76-81, which is incorporated herein by reference in its entirety;L282R, which is disclosed in Aldudo et al., Neurosci. Lett., 1998, 240,174-176, which is incorporated herein by reference in its entirety;G378A, which is disclosed in Besancon et al., Human Mutat., 1998, 11,481, which is incorporated herein by reference in its entirety; N405S,which is disclosed in Yasuda et al., J. Neurol. Neurosurg. Psychiatr.,2000, 68, 220-223, which is incorporated herein by reference in itsentirety; A409T, which is disclosed in Sugiyama et al., Online HumanMutat., 1999, 14, 90, which is incorporated herein by reference in itsentirety; L424R, which is disclosed in Kowalska et al., FoliaNeuropath., 1999, 37, 57-61, which is incorporated herein by referencein its entirety; a Delta exon 9 splice acceptor site deletion mutation(G

T with S290C), which is disclosed in Hardy et al., Trends Neurosci.,1997, 20, 154-159 and Lee et al., Nature Med., 1997, 3, 756-760, each ofwhich is incorporated herein by reference in its entirety; a Delta exon9 splice acceptor site deletion mutation (G

A with S290C), which is disclosed in Sato et al., Human Mutat. Supp.,1998, 1, S91-94, which is incorporated herein by reference in itsentirety; a Delta exon 9 Finn 4,555 basepair deletion, which isdisclosed in Prihar et al., Nature Med., 1999, 5, 1090, which isincorporated herein by reference in its entirety; a Delta intron 4splice donor consensus sequence G deletion, which is disclosed inDeJonghe et al., Human Molec. Genet., 1999, 8, 1529-1540, which isincorporated herein by reference in its entirety; and a C

T mutation at position −48 in the 5′ promoter, a C

G mutation at position −280 in the 5′ promoter, and an A

G mutation at position −2818 in the 5′ promoter, each of which isdisclosed in Theuns et al., Human Molec. Genet., 2000, 9, 325-331, whichis incorporated herein by reference in its entirety. Although theapplication exemplifies the P264L mutation in particular, all aspects ofthe invention can be applied to each and every human mutation recitedabove.

[0045] As used herein, “human P264L mutation” means the following: thenucleotide sequence of codon 264 of the presenilin 1 gene is changedfrom CCG to a sequence selected from the group consisting of: CTT; CTC;CTA; CTG; TTA; TTG; and most preferably changed from CCG to CTT.Additionally, the nucleotide sequence of codon 265 of the presenilin 1gene optionally, but preferably, is changed from AAA to AAG. The abovedescribed most preferable change of base sequences in codon 264constitute the human P264L mutation. The optional, but preferred, changeof the base sequence of codon 265 adds an AflII cleavage site to thegene.

[0046] As used herein, “target gene” or “targeted gene” means a gene ina cell, which gene is to be modified by homologous recombination with atargeting vector.

[0047] As used herein, “gene-targeted, non-human mammal” means anon-human mammal comprising one or more targeted genes via agene-targeting, as opposed to transgenic, approach.

[0048] As used herein, “generational offspring” in relationship to“gene-targeted, non-human mammal” means an animal whose genome includesthe same gene-targeted manipulation as the parent(s) of that offspring.For example, and not limitation, where a mammal whose genome has beenmanipulated by gene-targeting techniques to include a human mutation isthen used for breeding purposes, all subsequent generations derived fromthat first mammal(s) are considered “generational offspring” so long asthe genome(s) of such subsequent generational offspring comprises thegene-targeted manipulation as the original mammal; by design, thisdefinition does not exclude other genomic-manipulations which may alsobe present in such generational offspring, nor does this definitionrequire that such generational offspring be derived solely bycross-breeding techniques between a male and female mammal.

[0049] As used herein, “transgenic non-human mammal” means a non-humanmammal in which a foreign (“transgene”) gene sequence has been insertedrandomly in a non-human mammal's genome and is therefore expressed inaddition to all normally expressed native genes (unless the insertedtransgene has interrupted a gene thus preventing its expression).

[0050] As used herein, “targeting vector” or “replacement vector” meansa DNA molecule that includes arms of homology, the nucleotide sequence(located within or between the arms of homology) to be incorporated intothe target gene, and one or more selectable markers.

[0051] As used herein, “wild-type control animal” means anon-gene-targeted, non-human mammal of the same species as, andotherwise comparable to (e.g., similar age), a gene-targeted non-humanmammal as disclosed herein. A wild-type control animal can be used asthe basis for comparison, in assessing results associated with aparticular genotype.

[0052] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

[0053] The first step in producing a gene-targeted non-human mammal ofthis invention is to prepare a DNA targeting vector. The targetingvector is designed to replace, via homologous recombination, part of theendogenous presenilin 1 gene sequence of a non-human mammal, so as tointroduce the human mutation, preferably the P264L human mutation. Thetargeting vector is used to transfect a non-human mammalian cell, e.g.,a pluripotent, murine embryo-derived stem (“ES”) cell. In this cell,homologous recombination (i.e., the gene-targeting event) takes placebetween the targeting vector and the target gene. The mutant cell isthen used to produce intact non-human mammals (e.g., by aggregation ofmurine ES cells to mouse embryos) to generate germ-line chimeras. Thegermline chimeras are used to produce siblings heterozygous for themutated targeted gene. Finally, interbreeding of heterozygous siblingsyields non-human mammals (e.g., mice) homozygous for the mutated targetgene.

[0054] Targeting vectors for the practice of this invention can beconstructed using materials, information and processes known in the art.A general description of the targeting vector used in this inventionfollows.

[0055] A targeting vector or replacement vector for use in thisinvention has two essential functions: (1) to integrate specifically(and stably) at the endogenous presenilin 1 target gene; and (2) toreplace a portion of an exon of the endogenous presenilin 1 gene,thereby introducing the human mutation, and the mutation that introducesa new cleavage site in the gene. In a preferred embodiment, a portion ofexon 8 is replaced so as to introduce the P264L mutation. Those twoessential functions depend on two basic structural features of thetargeting vector.

[0056] The first basic structural feature of the targeting vector is apair of regions, known as “arms of homology,” which are homologous toselected regions of the endogenous presenilin 1 gene or regions flankingthe presenilin 1 gene. This homology causes at least part of thetargeting vector to integrate into the chromosome, replacing part (orall) of the presenilin 1 target gene, by homologous recombination.

[0057] Homologous recombination, in general, is the rearrangement of DNAsegments, at a sequence-specific site (or sites), within or between DNAmolecules, through base-pairing mechanisms. The present inventionrelates to a particular form of homologous recombination sometimes knownas “gene targeting.”

[0058] The second basic structural feature of the targeting vectorconsists of the actual base changes (mutation(s)) to be introduced intothe target gene. In the present invention, the base changes in codon 264of exon 8, for example, resulted in an amino acid change in amino acid264 from proline to leucine when the mutated gene was expressed to makeprotein. Other base changes can be made, as desired, to introduce any ofthe human mutations listed above into the mammalian genome. Themutation(s) to be introduced into the presenilin 1 target gene islocated within the “arms of homology.”

[0059] Gene targeting, which affects the structure of a specific genealready in a cell, is to be distinguished from other forms of stabletransformation, wherein integration of exogenous DNA for expression in atransformed cell is not site-specific, and thus does not predictablyaffect the structure of any particular gene already in the transformedcell. Furthermore, with the type of targeting vector preferred in thepractice of this invention (e.g., the one disclosed below), a reciprocalexchange of genomic DNA takes place (between the “arms of homology” andthe target gene), and chromosomal insertion of the entire vector isadvantageously avoided.

[0060] The examples of this patent disclosure set forth the constructionof a presenilin 1 gene targeting vector (and its use) to mutate themurine presenilin 1 protein encoding sequence so that it encodes themurine presenilin 1 protein, containing the human P264L mutation, or anyof the other human mutations recited above, and an additional cleavagesite. One of ordinary skill in the art will recognize that numerousother targeting vectors can be designed to introduce the same mutations,using the principles of homologous recombination. Gene-targeted,non-human- mammals produced using such other targeting vectors arewithin the scope of the present invention. A discussion of targetingvector considerations follows.

[0061] The length of the arms of homology that flank the replacementsequence can vary considerably without significant effect on thepractice of the invention. The arms of homology must be of sufficientlength for effective heteroduplex formation between one strand of thetargeting vector and one strand of a transfected cell's chromosome, atthe presenilin 1 target gene locus. Increasing the length of the arms ofhomology promotes heteroduplex formation and thus targeting efficiency.However, it will be appreciated that the incremental targetingefficiency accruing per additional homologous base pair eventuallydiminishes and is offset by practical difficulties in target vectorconstruction, as arms of homology exceed several thousand base pairs. Apreferred length for each arm of homology is 50 to 10,000 base pairs.

[0062] There is considerable latitude in the choice of which regions ofthe presenilin 1 target gene, i.e., chromosomal regions flanking thepresenilin 1 target gene, are represented in the targeting vector's armsof homology. The basic constraint is that the base pairs to be changedin the presenilin 1 target gene must lie within the sequence thatconstitutes the arms of homology. The arms of homology may lie withinthe presenilin 1 target gene, but it is not necessary that they do soand they may flank the presenilin 1 target gene.

[0063] Preferably, the targeting vector will comprise, between the armsof homology, a positive selection marker. The positive selection markershould be placed within an intron of the target gene, so that it will bespliced out of mRNA and avoid the expression of a target/marker fusionprotein. More preferably the targeting vector will comprise twoselection markers; a positive selection marker, located between the armsof homology, and a negative selection marker, located outside the armsof homology. The negative selection marker is a means of identifying andeliminating clones in which the targeting vector has been integratedinto the genome by random insertion instead of by homologousrecombination. Exemplary positive selection markers are neomycinphosphotransferase and hygromycin β phosphotransferase genes. Exemplarynegative selection markers are Herpes simplex thymidine kinase anddiphtheria toxin genes.

[0064] To eliminate potential interference on expression of the targetprotein, the positive selection marker can be flanked by short loxPrecombination sites isolated from bacteriophage P1 DNA. Recombinationbetween the two loxP sites at the targeted gene locus can be induced byintroduction of cre recombinase to the cells. This results in theelimination of the positive selection marker, leaving only one of thetwo short loxP sites. (See, U.S. Pat. No. 4,959,317, which is hereinincorporated by reference in its entirety). Excision of the positiveselectable marker from intron 8 of the mutated presenilin 1 gene canthus be effected.

[0065]FIG. 1 illustrates the general principles of gene-targeting forintroducing mutations into a mammalian genome using homologousrecombination (reviewed in Capecchi, M. R, Trends Genet., 1989, 5,70-76; Koller and Smithies, Ann. Rec. Immunol:, 1992, 10, 705-730). Alength of genomic DNA is first depicted by organizing it into regions(numbered 0-5 in FIG. 1a). In FIG. 1, several base pair changes (from 1-10) are to be incorporated into the cellular DNA around region 3.Homologous recombination using a gene targeting vector is utilized. Thetype of gene targeting vector used to incorporate these changes istermed a replacement vector.

[0066] As defined previously, a “replacement vector” herein refers to avector that includes one or more selectable marker sequences and twocontiguous sequences of ES cell genomic DNA that flank a selectablemarker. These flanking sequences are termed “arms of homology.” In FIG.1b, the arms of homology are represented by regions 1-2 and 3-4. The useof DNA derived from the ES cells (isogenic DNA) helps assure highefficiency recombination with the target sequences (te Riele et al.,Proc. Natl. Acad. Sci. USA, 1992, 89, 5128-5132). The arms of homologyare placed in the vector on either side of a DNA sequence encodingresistance to a drug toxic to the ES cells (positive selection marker).A gene encoding susceptibility to an otherwise nontoxic drug (negativeselection marker) is placed outside the region of homology. In thereplacement vector used in this invention, the positive selection markeris neo^(r), a gene that encodes resistance to the neomycin analog G418,and the negative selection marker is the herpes simplex virus thymidinekinase gene (HSV-tk) that encodes susceptibility to gancyclovir. Whenthis replacement vector is introduced into ES cells via transfection andits DNA undergoes homologous recombination with ES cellular DNA, thepositive selection marker is inserted into the genome between regions 2and 3 in this example (making the transformed cells resistant to G418)while the negative selection markers is excluded (making the cellsresistant to gancyclovir). Thus, to enrich for homologous recombinants,transfected ES cells are grown in culture medium containing G418 toselect for the presence of the neo^(r) gene and gancyclovir to selectfor the absence of the HSV-TK gene. Preferably, the positive selectionmarker is eliminated by using, for example, cre/lox technology once themammal is crossed with another mammal.

[0067] If base pair changes (mutations) are introduced into one of thearms of homology it is possible for these changes to be incorporatedinto the cellular gene as a result of homologous recombination. Whetheror not the mutations are incorporated into cellular DNA as a result ofhomologous recombination depends on where the crossover event takesplace in the arm of homology bearing the changes. For example, asdepicted by scenario “A” in FIG. 1, the crossover in the arm occursproximal to the mutations and so they are not incorporated into cellularDNA. In scenario “B”, the crossover takes place distal to the positionof the mutations and they are incorporated into cellular DNA. Becausethe location of the crossover event is random, the frequency ofhomologous recombination events that include the mutations is increasedif they are placed closer to the positive selection marker.

[0068] By the above method, the skilled artisan can achieve theincorporation of the selectable marker at a preselected location in thegene of interest flanked by specific base pair changes. Presumably, theartisan would preferably choose to have the selectable markerincorporated within the intron of the gene of interest so as not tointerfere with endogenous gene expression while the mutations would beincluded in adjacent coding sequence so as to make desired changes inthe protein product of interest (FIG. 1), (Askew et al., Mol. Cell.Biol., 1993, 13, 4115-4124, Fiering et al., Proc. Natl. Acad. Sci. USA,1993, 90, 8469-8473; Rubinstein et al., Nuc. Acid Res., 1993, 21,2613-2617, Gu et al., Cell, 1993, 73, 1155-1164, and Gu et al., Science,1994, 265, 103-106).

[0069] Thus, in the manner described above, a gene-targeted, non-humanmammal comprising a human PS-1 mutation is prepared. The mammal can beheterozygous (contains one copy of the human PS-1 mutation) orhomozygous (contains two copies of the human PS-1 mutation). In apreferred embodiment, a mouse is prepared which is PS-1^(P264L/+)(heterozygous) or PS-1^(P264L/P264L) (homozygous).

[0070] The gene-targeted, non-human mammals comprising a human PS-1mutation described above can be crossed with mammals having a SwedishFAD mutation and “humanized” Aβ sequence in the APP gene (e.g.,APP^(NLh/NLh) mouse) to produce mammals referred to asAPP^(Nlh/NLh)×PS-1^(P264L/P264L), APP^(NLh/+)×PS-1^(P264L/P264L),APP^(NLh/+)×PS-1^(P264L/+) or APP^(NLh/NLh)×PS-1^(P264L/+). In addition,the gene-targeted, non-hurman mammals comprising a human PS-1 mutationdescribed above can be crossed-with mammals having a Swedish APP695mutation (e.g., Tg2576 mouse). Prior to crossing such mammals, however,it is preferred to remove the positive selection marker, such asneo^(r), using cre/lox technology.

[0071] The present invention is also directed to a method foridentifiing a compound for treating Alzheimer's disease. A compound isadministered to a mammal that is heterozygous or homozygous for amutation of the PS-1 gene and also contains a human Swedish APP695mutation, or generational offspring thereof, or to a mammal heterozygousor homozygous for a mutation of the PS-1 gene, a human FAD Swedishmutation, and a humanized Aβ mutation, and generational offspringthereof. Any compound to be tested can be administered in a variety ofamounts by any variety of routes including, but not limited to,intravenously, orally, direct injection in the brain, and the like. Atissue sample from the mammal including, but not limited to, braintissue, non-brain tissue and body fluids (e.g. blood and plasma) isobtained and the amount of Aβ peptide in the tissue sample is measured.A decrease in the amount of Aβ peptide in the tissue sample isindicative of a compound that can be used to treat Alzheimer's disease.

[0072] The present invention is also directed to a method of treating anindividual suspected of having Alzheimer's disease. An individualsuspected of having Alzheimer's disease is any human having beenexamined by a physician and diagnosed as having Alzheimer's disease orsymptoms thereof. A compound identified by the methods described aboverelating to a mammal that is heterozygous or homozygous for a mutationof the PS-1 gene and also contains a human Swedish APP695 mutation, orgenerational offspring thereof, or to a mammal that is heterozygous orhomozygous for a mutation of the PS-1 gene, a human FAD Swedishmutation, and a humanized Aβ mutation, and generational offspringthereof, is administered to the individual in an amount effective todecrease the amount of Aβ peptide in the brain of the individual. Anamount effective to decrease the amount of Aβ peptide can be determinedfrom the identification process of the compound using a mammal that isheterozygous or homozygous for a mutation of the PS-1 gene and alsocontains a human Swedish APP695 mutation, or generational offspringthereof, or using a mammal that is heterozygous or homozygous for amutation of the PS-1 gene, a human FAD Swedish mutation, and a humanizedAβ mutation, or generational offspring thereof, as a starting amount andscaling up for use in humans as is well known to those skilled in theart. An effective Alzheimer's disease treatment amount is an amount of acompound that measurably reduces the physiological pathology ofAlzheimer's disease or an amount that reduces the physicalmanifestations or symptoms of Alzheimer's disease. One skilled in theart can, for example, begin with an amount of a compound that decreasesthe amount of Aβ peptide in the brain, as described above, and can scaleup or down the amount depending on the desired effect and the effectachieved in a particular individual.

[0073] The present invention is also directed to compounds that areidentified by the screening methods described above. The compounds canbe any identifiable chemical or molecule, including, but not limited to,a small molecule, a peptide, a protein, a sugar, a nucleotide, or anucleic acid, and such compound can be natural or synthetic.

[0074] In order that the invention disclosed herein may be moreefficiently understood, examples are provided below. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting to the invention in any manner.Throughout these examples, molecular cloning reactions, and otherstandard recombinant DNA techniques, were carried out according tomethods described in Maniatis et al., Molecular Cloning-A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor Press (1989) (hereinafter,“Maniatis et al.”), using commercially available enzymes, except whereotherwise noted.

EXAMPLES Example 1 Cloning of Mouse PS-1 Exon 8 Region

[0075] The mouse PS-1 genomic DNA was cloned from a bacteriophagelibrary created from 129/Sv mouse DNA partially digested with Sau3A andinto the BamHI site of Lambda DASH™ II (Reaume et al., Science, 1995,267, 1831-1833, which is incorporated herein by reference in itsentirety). Using standard molecular biology techniques (Maniatis etal.), approximately 1.2×10⁶ recombinant bacteriophages were screened forthe presence of PS-1 sequences by hybridization with a small,radiolabeled PS-1 specific DNA probe. This 477 base pair PS-1 probe wasgenerated by polymerase chain reaction (PCR) amplification (Mullis etal., Methods Enzymol., 1987, 155, 335-350) of mouse genomic DNA usingprimers R892 (CTC ATC TTG GCT GTG ATT TCA; SEQ ID NO:1) and R885 (GTTGTG TTC CAG TCT CCA; SEQ ID NO:2) which hybridize to the 3′ end of exon7 and the 5′ end of exon 11 respectively (FIG. 2). The amplifiedfragment was separated from other components of the reaction byelectrophoresis on a 1.0% agarose gel, and purified using GeneClean®II(Bio 101, Inc., La Jolla, Calif.). Purified probe DNA was radioactivelylabeled with ³²P-dCTP by the random primer method using materials andmethods supplied by the kit manufacturer (Multiprime DNA LabelingSystem; Amersham Life Sciences, Arlington Heights, Ill.).

[0076] From this screen, 13 clones were identified that hybridized tothe PS-1 probe. The clones were identified as: λPS1-4, λPS1-5, λPS1-6,λPS1-10, λPS1-11, λPS1-17, λPS1-19, λPS1-20, λPS1-22, λPS1-24, λPS1-28,λPS1-31,and λPS1-35. These clones were purified by limiting dilution andplaque hybridization with the PS-1 probe (Maniatis et al.).

[0077] From each clone, DNA was prepared from bacteriophage particlespurified on a CsCl gradient (Maniatis et al.). Restriction maps werethen generated for each of the cloned inserts using the FLASH™Non-radioactive Gene Mapping Kit (Stratagene® Inc., La Jolla, Calif.). Atypical restriction map generated by this method is illustrated in FIG.3. This method of restriction enzyme mapping involves first completelydigesting 10 μg of the bacteriophage DNA with the restriction enzymeNotl using standard restriction enzyme digest conditions (Maniatis etaL) . Notl cuts all clones in the vector DNA at either end of the clonedinsert so as to leave a T3 bacteriophage promoter attached to one end ofthe insert and a T7 bacteriophage promoter attached to the other end.The Notl digested DNA is then partially digested with the enzyme EcoRl,as an example, using limiting amounts of enzyme (0.2 units/μg DNA) in an84 μl reaction volume at 37° C. Aliquots (26 μl) were removed after 3minutes, 12 minutes and 40 minutes and the digest reaction was stoppedby the addition of 1 μl of 0.5 M EDTA. DNA from all three time pointswas resolved on a 0.7% agarose gel, visualized by ethidium bromidestaining, and then transferred to a GeneScreen Plus® membrane (NEN®Research Products, Boston, Mass.) by capillary transfer (Maniatis etal., supra). The membrane was hybridized with an alkaline phosphataselabeled oligonucleotide that was specific for the T3 promoter (suppliedwith the FLASH™ kit) using reagents and methods supplied by the kitmanufacturer. After hybridization, the membrane was washed and developedwith a chemiluminescent-yielding substrate and then exposed to X-rayfilm in the dark for approximately 60 minutes.

[0078] The oligonucleotide probes effectively label one end of theinsert. By determining the positions of the bands on the X-ray film andcalculating the DNA size to which they corresponded, it was possible todetermine the position of the EcoRl sites relative to the T3 end of theinsert (FIG. 3). These results could then be complemented by strippingthe probe off of the membrane, and rehybridizing with a T7-specificoligonucleotide in order to determine the positions of the EcoRl sitesrelative to the T7 end of the insert. This process was repeated usingthe enzymes HindIII and XbaI. By comparing the restriction enzyme mapsof the different overlapping clones a composite map was assembled. Ofthe 13 original clones isolated, 6 independent clones were identified(FIG. 2).

[0079] Exon 8 was located on the restriction map hybridizingexon-specific probes to complete digests of each of the 6 differentlambda genomic clones. Initially, 3 μg of DNA from each of the 6different clones was completely digested with the restriction enzymesEcoRl and Xbal. The digested DNA was resolved on a 0.8% agarose gel,visualized by means of ethidium bromide staining and transferred to aGeneScreen Plus® membrane by capillary transfer. The membrane was thenhybridized with a DNA probe that specifically hybridized to sequencesfrom mouse PS-1 exon 8. This probe was generated by PCR usingoligonucleotides FEX8 (ATT TAG TGG CTG TTT TGT G; SEQ ID NO:3) and REX8(AGG AGT AAA TGA GAG CTG GA; SEQ ID NO:4) which hybridize to the 5′ and3′ ends of exon 8, respectively. After hybridization, the membrane waswashed and exposed to X-ray film (FIG. 4). This experiment revealed thatall clones contained a 2.5 kb fragment that hybridized to the exon 8probe. By combining this information with the restriction map data foreach lambda clone, exon 8 was identified on the map (position 11.5 to 14on the summary map, FIG. 2).

[0080] A similar procedure was used to identify the position of exon 7on our composite map using exon 7-specific probe and utilizing therestriction enzymes Xbal and EcoRl. The exon 7-specific probe wasgenerated using PCR primers F892 (TGA AAT CAC AGC CAA GAT GAG; SEQ IDNO:5) and PS1-1 (GCA CTC CTG ATC TGG AAT TTT G; SEQ ID NO:6). Exon 7 waslocalized to the 2 kb Xbal-EcoRl fragment of all clones except λPS1-22which allowed for the determination that exon 7 is located betweenpositions 7.0 and 9.0 on the summary map (FIG. 2).

[0081] Exon-specific probes were also used to obtain additionalrestriction map information using additional restriction enzymes. Forexample, when λPS1-22 was digested with Notl and BamHI, resolved on anagarose gel, transferred to a Genescreen Plus® membrane and probed withthe exon 8-specific probe, a 700 bp fragment was identified. Thisinformation, when combined with the information from the otherbacteriophage clones, allowed placement of the BamHI at position 11.7 onthe composite map (FIG. 2). This process was repeated for therestriction enzyme Xhol.

[0082] Cloning of additional regions of the mouse PS-1 gene can also beaccomplished, as desired, in order to prepare additional vectorscomprising other human mutations.

Example 2 Construction of a Replacement Vector

[0083] A 4.7 kb XbaI-BamHI fragment (which also contains two internalXbaI fragments) located at positions 7.0 to 11.7 on the summary map(FIG. 2), was chosen as the 5′ arm that included the necessary mutationsand a 4.1 kb BamHI-EcoRI fragment (which also contains an internal EcoRIsite) located at positions 11.7 to 15.8 on the summary map (FIG. 2), asa 3′ arm. These fragments were isolated first and cloned intopBlueScript® SK+ (Stratagene Cloning Systems, LaJolla, Calif.) and thenfurther subcloned into the plasmid pPNTIox² (described below) thatcontained a neo^(r) gene, an HSV-TK gene and linker sequences to producea replacement vector (pPS1-8-TV, FIG. 5) with the same general structureas that discussed above.

[0084] (a) Construction of the Intermediate Plasmid pPNTlox²

[0085] pPSI-8-TV was created from pPNT (Tybulewicz et al., Cell, 1991,65, 1153-1163; obtained from Dr. Richard Mulligan, MIT) by firstinserting two oligonucleotide linkers on each side of the neo^(r)cassette creating the intermediate called pPNTIox² (FIG. 6). Adouble-stranded 79 base pair 5′ linker was created by annealing twosingle-stranded oligonucleotides that overlap at their 3′ ends and thenfilling in the remaining single-stranded regions with the Klenowfragment of DNA polymerase I. The oligonucleotides PNT Not (GGA AAG AATGCG GCC GCT GTC GAC GTT AAC ATG CAT ATA ACT TCG TAT; SEQ ID NO:7) andPNT Xho (GCT CTC GAG ATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA TAT GC;SEQ ID NO:8) (150 ng of each) were combined in a 30 μl reaction mixturecontaining 5 U of Klenow polymerase, Klenow polymerase buffer and 2 mMdNTPs (dATP, dCTP, dGTP, and dTTP). After incubating for 1 hour at 37°C., a portion (5 μl) of this reaction mixture was simultaneouslydigested with the restriction enzymes NotI and XhoI to liberate therestriction enzyme sites at each end of the linker. In addition, 200 ngof pPNT was digested with NotI and XhoI. The digested plasmid wasresolved on a 0.8% agarose gel, purified from the gel, and treated withcalf intestinal phosphatase according to standard methods (Maniatis etal.). A quantity (66 ng) of the double digested linker was ligated tothe double-digested and phosphatase-treated pPNT DNA (Maniatis et al.).Following DNA transformation of competent WM 1100 E. coli (Dower,Nucleic Acids Res., 1988, 16, 6127-6145), plasmid DNA was isolated fromampicillin-resistant bacteria (Holmes et al., Anal. Biochem., 1981, 114,193-197) and analyzed by restriction enzyme analysis. The properrecombinant plasmids were identified as having acquired SalI, HpaI andNsiI sites (present in the linker) while still retaining the NotI andXhoI sites of the starting plasmid. One such recombinant plasmid with a79 bp linker sequence was identified and called pXN-4 (FIG. 6).

[0086] A similar approach was used to insert a 3′ linker between theXbaI and BamHI sites of pXN-4. The oligonucleotides used to synthesizethe linker were PNT Xba (CGT TCT AGA ATA ACT TCG TAT AAT GTA TGC TAT;SEQ ID NO:9) and PNT Bam (CGT GGA TCC ATA ACT TCG TAT AGC ATA CAT TAT;SEQ ID NO:10). In this case, pXN-4 and the double-stranded linker DNAwere digested with XbaI and BamHI. The purified fragments were joined byDNA ligation and transformed into competent WM1100 bacteria. Plasmid DNAwas digested with XbaI and BamHI, end-labeled with ³²P-dCTP and Klenowpolymerase, and resolved on an 8% acrylamide gel (Maniatis et al.). Thegel was dried and exposed to X-ray film. Proper recombinant clones wereidentified by the presence of a 40 base pair band liberated by theXbaI-BamHI double digest. The resulting plasmid was designated“pPNTIox²” (FIG. 6).

[0087] To confirm the sequences of the inserted linkers, a fragmentcontaining both linkers was isolated from pPNTIox² using NotI and EcoRIand cloned into pBlueScript® SK+, a vector that was more amenable tonucleotide sequencing. Identity of the linkers was confirmed by directnucleotide sequencing (Sanger, Proc. Natl. Acad. Sci. USA, 1977, 74,5463-5467) using T3 and T7 sequencing primers (Stratagene® Inc., LaJolla, Calif.) and Sequenase Version 2.0 DNA Sequencing Kit (UnitedStates Biochemical, Cleveland, Ohio).

[0088] (b) Subcloning Arms of Homology.

[0089] An XbaI-HindIII fragment (positions 11.5 to 15.9 on the summarymap, FIG. 2) containing the 3′ arm of homology and the fragment used forin vitro mutagenesis was first isolated from λPS1-22 by digesting 30 μlof the phage DNA with XbaI and HindIII, resolving the digested DNA on a0.8 agarose gel, visualizing the DNA with ethidium bromide staining andthen excising the 4.4 kb fragment from the gel. DNA was purified fromthe gel using GeneClean® II (Bio101 Inc., La Jolla, Calif.).Simultaneously, 1 μg of pBlueScript® SK+ was digested with XbaI andHindIII and subsequently purified by the same procedure. Approximately400 ng of the purified lambda DNA and 100 ng of the purified plasmid DNAwere combined in a 10 μl ligation reaction. Following taansformation ofcompetent WM100 E. coli, plasmid DNA was isolated fromampicillin-resistant bacteria and analyzed by restriction enzymeanalysis to identify the resultant plasmids (FIG. 7). In this case,plasmid DNA from transformed bacteria was first analyzed by digesting itwith XbaI and HindIII in order to determine whether the plasmid DNA hadacquired the 4.4 kb PS-1 fragment. This plasmid was designated “pPSI-XH16.”

[0090] Similar procedures were used to isolate a 200 bp XbaI-BamHIfragment from pPS1-XH16 and subclone it into pBlueScript® SK+. Theresulting plasmid was designated “pPS1-XB1” (FIG. 8).

[0091] One of the fragments in the 5′ arm of homology (a 4.2 kb XbaIfragment at positions 7.0 to 11.2 on summary map; FIG. 2) was similarlysubcloned from λPSI-6 into pBlueScript® SK+ and designated “pPS1-X15”(FIG. 9). Because this insert could be positioned in the plasmid ineither of two orientations, plasmid DNA was further screened bydigesting it with the enzyme EcoRI. In this way, it was determined thatthe clone pPS1-X15 had the PS-1 insert oriented such that the 5′ end wasclosest to the T3 promoter while in pPS1-X2 the 5′ end was adjacent tothe T7 promoter (FIG. 9).

[0092] The 300 bp XbaI fragment in the 5′ arm (position 11.2 to 11.5 onsummary map; FIG. 2) was also similarly cloned into pBlueScript® SK+from λPS1-20 and named pPS1-X319 (FIG. 10). In this case, theorientation of the XbaI fragment was not determined by subsequentrestriction mapping.

[0093] (c) Restriction Mapping Arms of Homology.

[0094] Further restriction enzyme mapping was performed on the pPS1-X315and pPS1-X2 As an example, each of the two plasmids were digested withthe enzyme HincII, resolved on an agarose gel, and visualized withethidium bromide. Because a HincII site is known to exist in thepBlueScrip® SK+ plasmid backbone within the multiple cloning site regionnear the T7 promoter relative to the insert position, it was possible todetermine the position of the HincII site in the 4.2 PS-1 fragment bydetermining the fragment sizes in each of the two digested samples (FIG.11).

[0095] Positions of restriction enzymes sites that occurred once ortwice in the 4.2 kb PS-1 fragment were determined by the above method.If more than two sites of a given enzyme were present, it becamenecessary to determine the relative positions by double-digesting eachof the two plasmids with the enzyme in question as well as an additionalenzyme which cut at sites capable of resolving ambiguities. In manycases, enzymes that cut more than twice were not resolved in this waybut simply noted as having multiples sites in the 4.2 kb PS-1 fragment.The list of additional enzymes used to characterize this region include,but are not limited to, AccI, ApaI, BamHI, EcoRI, HincII, HpaI, KpnI,NsiI, PstI, SaII, SmaI, SpeI, and XhoI. A summary of these data isillustrated in FIG. 12. The same procedures were used to create arestriction enzyme map for the pPS1-XH16 (FIG. 12).

[0096] (d) Mutagenesis of the 3′ arm of homology.

[0097] A total of 3 base pair changes were introduced into the exon 8region using a PCR strategy (for summary of changes, see FIG. 13). TheP264L mutation, and an AflII site were introduced. Teri ng of pPS1-XB1were included into each of two PCR reactions. The first reactioncontained the primers EXPL2 (TTG TGT CTT AAG GGT CCG CTT CGT ATG; SEQ IDNO:1 1) and T7 (Stratagene Cloning Systems, La Jolla, Calif.). Thisgenerated a 220 bp band that encompassed the 3′ end of exon 8 and clonePS1-XB1. This fragment also included the P264L mutation and a novelAflII site that resulted as part of the P264L change.

[0098] The second PCR reaction used the primers EXPL1 (CGG ACC CTT AAGACA CAA AAC AGC CAC; SEQ ID NO:12) and T3 (Stratagene Cloning Systems,La Jolla, Calif.). This generated a 137 bp fragment that encompassed the5′ end of exon 8 and PS1-XB1. This fragment also included the P264Lchange and an AflII site (FIG. 14).

[0099] The product of the first reaction was purified using Magic™ PCRPreps DNA Purification System (Promega Corporation, Madison, Wis.) anddigested with BamHI and AflII in order to liberate the restriction sitesat its ends. Similarly, the product of the second reaction was purifiedand digested with AflII and XbaI. These two fragments, as well as XbaIand BamHI digested pBlueScript® SK+ were ligated together andtransformed into HB101 competent E. coli cells. The DNA was isolated andanalyzed from the ampicillin resistant colonies. The clone bearing arecombinant plasmid in which the two PCR fragments had joined togetherat their AflII site and inserted into the BamHI and XbaI sites ofpBlueScript® SK+ was called pPS1-XB85 (FIG. 14). To confirm thesequences of the mutagenized exon 8, direct nucleotide sequencing(Sanger, Proc. Natl. Acad Sci. USA, 1977, 74, 5463-5467) was performedusing T3 and T7 sequencing primers (Stratagene Inc., LaJolla, Calif.)and Sequenase Version 2.0 DNA Sequencing Kit (United States Biochemical,Cleveland, Ohio).

[0100] The 5′ arm of homology was assembled in pBlueScript® SK+ throughseveral cloning steps. First, pPS1-XB15 was partially digested with XbaIso that only one XbaI site was cut. The resulting DNA was then digestedwith BamHI and gel purified (FIG. 15).

[0101] The mutated insert in pPS1-XB85 was released by digesting it withXbaI and BamHI and gel purifying the resulting mutated insert. The 200bp XbaI-BamHI fragment was ligated into the linearized pPS1-X15 andrecombinant plasmids were screened for the proper orientation of theinsert by means of an AflII digest. The correctly oriented plasmidyielded 1.9 kb and 5.8 kb fragments. This plasmid was designated“pPS1-206.”

[0102] To insert the small 300 bp XbaI fragment 5′ relative to themutated 200 bp XbaI-BamHI fragment, pPS1-206 was linearized by a partialXbaI digest (FIG. 16). The XbaI fragment from pPS1-X319 was isolated andcloned into the linearized pPS1-206 DNA. Orientation of the 300 bp XbaIfragment was determined by sequencing the recombinant clone as well asλPS1-20 with primer EX8PL1 using the Thermo Sequenase radiolabeledterminator cycle sequencing kit (Amersham Life Science Inc., Cleveland,Ohio). A plasmid clone that shared sequence identity with λPS1-20 beyondthe XbaI site had the 300 bp XbaI fragment inserted in the properorientation. This plasmid, which contained the assembled 5′ arm, wasdesignated “pPS1 -5′360” (FIG. 16).

[0103] (e) Assembling the Targeting Vector pPS-1-8-TV.

[0104] The plasmid pPNTlox² was prepared for receiving the 3′ arm ofhomology by first digesting plasmid DNA with EcoRI and BamHI and gelisolating the linear plasmid (FIG. 17). In parallel, the 3′ arm wasprepared by partially digesting pPS1-XH16 with EcoRI and isolating thelinear form. This fragment was then digested with BamHI and the 4.1 bpfragment gel isolated. The 3′ arm was ligated to pPNTlox². The resultingplasmid was designated “pPNT3′413.”

[0105] The 5′ arm was inserted into pPNT3′413 to give the final plasmidpPSI-8-TV. The 5′ arm was liberated from plasmid DNA by first digestingwith XhoI and NotI. In parallel, pPNT3′413 was prepared by doubledigesting with Noti and SalI The two fragments of DNA were ligated andtransformed into competent WM 1100 E. coli cells (FIG. 18).

[0106] Additional vectors can be prepared in the manner described abovein order to comprise other human mutations.

Example 3 Mutagenesis of the Mouse PS-1 Gene in ES cells

[0107] (a) Cells.

[0108] The R1 line of ES cells derived from 129/Sv×129/Sv-CP FI hybridmice (Nagy et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 8424-8429) andobtained from Dr. Janet Rossant (Mt. Sinai Hospital, Toronto, Ontario,Canada) was utilized. These cells were grown in ES cell mediumconsisting of Dulbecco's Modification of Eagle's Medium (withL-glutamine and 4500 mg/L glucose; Mediatech Inc., Herndon, Va.)supplemented with 20% fetal bovine serum (FBS; Hyclone LaboratoriesInc., Logan, Utah; cat. # A-1115; Lot # 11152154), 0.1 mM non-essentialamino acids (Mediatech 25-025-L1), 2 mM L-glutamine (Mediatech25-005-L1), 10⁻⁶ M β-mercaptoethanol (Gibco 21985-023) 1 mM sodiumpyruvate (Mediatech 25-000-L1), 1× concentration of a penicillinstreptomycin solution (Mediatech 30-001-L1) and 1000 U/ml of leukemiainhibitory factor (Gibco BRL 13275-029). The cells were grown on tissueculture plastic that had been briefly treated with a solution of 0.1%gelatin (Sigma G9391).

[0109] The cultures were passed every 48 hours or when the cells becameabout 80% confluent. For passage, the cells were first washed withphosphate buffered saline (without Ca²⁺ and Mg²⁺) and then treated witha trypsin/EDTA solution (0.05% trypsin, 0.02% EDTA in PBS without Ca²⁺and Mg²⁺). After all of the cells were in suspension, the trypsindigestion was stopped by the addition of tissue culture medium. Thecells were collected by centrifugation, resuspended in 5 ml of tissueculture medium and a 1 ml aliquot of the cell suspension was used tostart a new plate of the same size.

[0110] (b) DNA Transfection of ES Cells.

[0111] pPS1-8-TV DNA (400 μg) was prepared for electroporation bydigesting it with NotI in a 1 ml reaction volume. The DNA was thenprecipitated by the addition of ethanol, washed with 70% ethanol andresuspended in 500 μl of sterile water.

[0112] The NotI-linearized pPS1-8-TV DNA was electroporated into EScells using a Bio-Rad Gene Pulser® System (Bio-Rad Laboratories,Hercules, Calif.). In each of 10 electroporation cuvettes, 40 μg of DNAwas electroporated into 2.5×10⁶ cells suspended in ES cell culturemedium. The electroporation conditions were 250 V and 500 μF whichtypically result in time constants ranging between 6.0-6.1 seconds.After electroporation the cells were incubated for 20 minutes at roomtemperature in the electroporation cuvettes. All the electroporatedcells were then pooled and distributed equally onto 10 gelatinizedplates. After 24 hours the plates were aspirated and fresh ES cellmedium was added. The next day, the medium in 9 plates was replaced withES cell medium supplemented with 150 μg/ml of G418 (Gibco) and 0.2 μMgancyclovir (Syntex) while one plate received medium supplemented onlywith 150 μg/ml of G418. After an additional 8 days, resulting individualES cell colonies were picked off of the plates and separately expandedin a well of 24-well plates as described by Wurst et al., pp 33-61 inGene Targeting Vol. 126, 1993, Edited by A. L. Joyner, IRL Press, OxfordUniversity Press, Oxford, England. Comparison of the number of coloniesthat grew on the plates supplemented with G418 and gancyclovir versusthe number that grew with only G418 supplementation was used todetermine the efficiency of negative selection.

[0113] (c) Analyses of the ES Cell Transformants.

[0114] When the cell culture in each well of the 24-well plates becameapproximately 80% confluent, it was washed and the cells were dispersedwith two drops of trypsin-EDTA. Trypsinization was stopped by theaddition of 1 ml of ES cell medium. An aliquot (0.5 ml) of thissuspension was transferred to each of two wells of separate 24-welplates. After the cells had grown to near confluence, one of the plateswas used for cryopreservation of the cell line while the other was usedas a source of DNA for each of the clones.

[0115] For cryopreservation, the cells in a 24-well plate were firstchilled by placing the plate on ice. The medium was then replaced withfresh ES cell medium supplemented with 10% DMSO and 25% FBS and theplate was cooled at approximately 0.5° C./min by insulating the plate ina styrofoam box and placing it in a −70° C. freezer.

[0116] To isolate the DNA from the clones on the other plate, the mediumin each well was replaced with 500 μl of digestion buffer (100 mMTris-HCl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, 100 μg/ml proteinaseK). After overnight incubation at 37° C., 500 μl of isopropanol wasadded to each well and the plate was agitated for 15 minutes on anorbital shaker. The supernatant was aspirated and replaced with 500 μlof 70% ethanol and the plate was shaken for an additional 15 minutes.The DNA precipitate was picked out of the well and dissolved in 50 μl ofTE solution (10 mM Tris-HCl pH 7.5, 1 mM EDTA).

[0117] The primary analysis for mutagenesis of the mouse PS-1 geneinvolved a Southern hybridization screen of ApaI digested ES cell DNA.The probe for this analysis was derived from the 3′ end of our clonedPS-1 region outside of the 3′ arm of homology (FIG. 19d). It wasprepared by f isolating the 6 kb XbaI fragment corresponding to the 3′end of λPS1-6 (FIG. 2) and subcloning it into XbaI digested pBlueScript®SK+. A further digest of this subclone, called pPS1-X6 with Xhol (aninternal site) and HindIII (from the Bluescript® S K+ polylinker)yielded the 1000 bp probe.

[0118] For the Southern hybridization screen, an aliquot (10 μl) of eachES cell clone DNA was digested with ApaI, resolved on a 0.8% agarosegel, and transferred to a GeneScreen Plus® membrane. The probe waslabelled with ³²P-DCTP by random priming and hybridized overnight to themembrane at 58° C. (Church et al., Proc. Natl. Acad Sci, USA, 1984, 81,1991-1995). An ES cell line in which the PS-1 gene has successfullyundergone homologous recombination yields 9 and 15 kb Apal fragments inthis assay (FIG. 19). This is because homologous recombinationadvantageously introduces a novel ApaI site into the region where theneo^(r) cassette is incorporated. The 15 kb band represents theunaltered cellular copy of PS-1 while the 9 kb band is derived from thePS-1 copy in which the novel ApaI site results in a shorter fragment. Inthis first screen, 8 cell lines were identified as potential targetedcell lines out of 260 cell lines analyzed.

[0119] All cell lines scored as putative homologous recombinants by theprimary screen were then further screened using a 500 bp KpnI-ApaIfragment isolated from a 5.5 kb 5′ XbaI fragment from λPS1-20 on ScaIdigested ES cell DNA. In this case, the normal PS-1 gene yielded a 13.8kb fragment and the mutant PS-1 gene a 10.5 kb fragment (FIG. 19e). Ofthe 8 cell lines examined in this screen, 4 were shown to have undergonehomologous recombination at their 5′ end.

[0120] Cell lines that were identified as having undergone homologousrecombination by both screens were considered to have undergone bonafidehomologous recombination (as opposed to homologous insertion which wouldgive positive results for only 1 of the 2 preceding screens). However,depending on where crossover takes place when the 5′ arm recombines, themutations that were included in this arm may or may not have beenincorporated into cellular DNA as a result of proper homologousrecombination (FIG. 1). A further Southern hybridization screen aimed atdetecting the novel AflII site created as a result of the P264L mutationwas therefore implemented. For this, a 1.2 kb HindIII-XbaI fragmentisolated from pPS1-X15 as a probe on AflII digested DNA was utilized. Anunaltered PS-1 gene yielded a 6.7 kb band (FIG. 19f). A PS-1 gene inwhich proper homologous recombination has taken place, but which lacksthe planned mutations yields a 8.7 kb band while the inclusion of theplanned mutations yields a 2.2 kb band. Of the 4 bona fide homologousrecombinant cell lines examined, all 4 were shown to have incorporatedthe novel AflII site near the planned mutations.

[0121] The mutagenized form of the PS-1 gene described here has beencalled PS1^(nP264L) as opposed to the normal PS-1 gene termed PSI⁺. Thefour ES cell lines bearing one copy of PS1^(nP264L) have been called,PS1-87, PS1-175, PS1-176, and PS1-243. Three of these lines were thawed,cell numbers expanded, and used to establish PS-1 mutant mice.

[0122] Additional mutagenesis of the mouse PS-1 gene can be performed inES cells in the manner described above in order to comprise other humanmutations.

Example 4 Establishment of PS-1 Mutant Mice

[0123] PS-1 mutant ES cells were used to make chimeric mice byaggregating the mutant ES cells to E2.5 embryos and transferring theaggregated embryos to pseudopregnant females (Wood et al., Nature, 1993,365, 87-89). ES cells were prepared for aggregation by limitedtrypsinization to produce clumps that average 10-15 cells. E2.5 embryoswere collected from superovulated CD-1 female mice by oviduct flushingas described by Hogan et al., Manipulating the Mouse Embryo: ALaboratory Manual, 1986, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). The zona pelucida was removed from the embryos usingacidic Tyrode's solution (Sigma Chemical Co., St. Louis, Mo.).Aggregation wells were created by pressing a blunt metal instrument (adarning needle) into tissue culture plastic. Embryos were then placed ina well together with a clump of approximately 10-15 ES cells in a smalldrop (approximately 20 μl) of M16 medium (Sigma Chemical Co., St. Louis,Mo.) under mineral oil. After an overnight incubation (37° C., 100%humidity, 5% CO₂ in air) the aggregate embryos were transferred to theuterine horns of a pseudopregnant female (Hogan et al., 1986, supra).Contribution of the ES cells to the offspring was scored by theappearance of pigmented coat color. Positive mice are termed chimericfounders. Germline contribution by the ES cells was scored by theappearance of pigmented offspring from a cross between the chimericfounders and CD-1 females.

Example 5 Chimeras

[0124] Of 3 mutant PS-1 ES cell lines used in embryo aggregations, oneproduced a germline chimera: TABLE 1 Number Number Number of Embryo ofChimeric of Germline Clone Aggregation Founders Chimeras PSI-175 400 5 1PSI-176 75 4 0 PSI-243 120 0 0

[0125] The germline chimera was then used to establish lines of micecarrying PS-1^(nP264L). The presence of the mutant PS-1 allele in thepigmented offspring was determined using a PCR strategy aimed atdetecting the neo^(r) cassette, following substantially the sameprocedure as set forth in Example 1. PCR primers were as follows: neo28(GGA TTG CAC GCA GGT TCT CC; SEQ ID NO:13); and neo445 (CCG GCT TCC ATCCGA GTA CG; SEQ ID NO:14). The genomic DNA was prepared from a tailsample (Hogan, 1986, supra). Of the four pigmented offspring, one femalemouse was heterozygous for PS-1^(nP264L) (PS-1^(nP264L/+)), i.e., thismouse was positive for the neo^(r) cassette based upon the foregoing PCRstrategy. Subsequent generational offspring which are also heterozygousfor PS-1^(nP264L) have been developed by mating of this female withwild-type males.

[0126] Mice heterozygous for PS-1^(nP264L) (PS-1^(nP264L/+)) weregenotyped using a PCR-based method. The presence of the wild-type allelefor murine PS-1 was scored using the following primers: X8F (CCC GTG GAGGAG GTC AGA AGT CAG; SEQ ID NO:15) and X8R (TTA CGG GTT GAG CCA TGA ATG;SEQ ID NO:16). Scoring with these primers yields a 142 bp fragment (datanot shown). The presence of the mutant allele was scored using theprimers neo28 and neo445, which yields a 417 bp fragment. Thus, micewhich are heterozygous for the mutation yield both bands; mice which arehomozygous for the mutation yield only the 417 bp band; and mice thatare homozygous for the wild-type allele yield only the 142 bp band.Tissue samples were derived from animal tails, and the PCR procedures ofExample 1 were utilized for such scoring.

[0127] Mice homozygous for the PS-1^(nP264L) allele (i.e.,PS-1^(nP264L/nP264L)) were generated by cross breeding of heterozygousmice (PS-1^(nP264L/+)) with mice which are homozygous for a humanizedAPP gene (as disclosed in PCT Publication Number W096/34097, publishedOct. 31, 1996; incorporated herein fully by reference). The resultinggenerational offspring were then determined to be heterozygous for boththe PS-1^(nP264L) allele and heterozygous for the humanized APP gene(data not shown); these generational offspring were then utilized forcross-breeding, with resulting generational offspring determined (usingthe PCR procedure outlined above) to be homozygous for the PS-1^(nP264L)allele, as well heterozygous for the humanized APP gene (generationaloffspring from this liter were also found to be heterozygous for thePS-1^(nP264L) allele/homozygous for the humanized APP gene; andheterozygous for the PS-1^(nP264L) allele/heterozygous for the humanizedAPP gene—due to the limited number of pups obtained from this liter,double homozygotes were not found). Subsequent matings producedPS1^(nP264L/nP264L)×APP^(NLh/NLh) mice.

[0128] Mice homozygous for the PS-1^(nP264L) allele were also generatedby cross-breeding of heterozygous mice (PS-1^(nP264L/+)). In one set ofmatings, 6 homozygotes were found amongst 27 offspring, which is wellwithin the expected 25% recovery of homozygotes from a heterozygouscross.

[0129] Accordingly, and based upon the various breeding approachesdisclosed above, substantially normal viability and embryonic survivalof the animals is evident.

Example 6 Excision of the PGK-neo Cassette

[0130] pBS185 plasmid DNA (Sauer et al., New Biol., 1990, 2, 441-449,incorporated herein by reference in its entirety) encoding Crerecombinase was introduced by pronuclear injection into one-cell embryosgenerated from a PS-1^(nP264L/+)×CD-1 cross. Since the plasmid wascircular, DNA integration into the genome had a very low frequency ofoccurrence. Transient expression of the DNA to produce Cre recombinaseexcised the PGK-neo cassette in the early embryos. Injected embryos weretransferred to pseudopregnant females. Excision of the PGK-neo cassettewas confirmied by genotyping of the offspring. These mice weredesignated PS-1^(P264L/+) and were crossed to generatePS-1^(P264L/P264L) mice.

[0131] Because the neomycin-selectable marker reduced transcription ofthe PS-1 gene, the PGK-neo gene was excised by recombination at theflanking loxP sites after transient expression of Cre recombinase.One-cell embryos (n=154) generated by a PS-1^(nP264L/+)×CD-1 cross wereinjected with pBS185 plasmid DNA and implanted into pseudopregnantfemales. The loss of the PGK-neo gene was scored in the offspring as a219-base pair fragment by PCR using the X8F and X8R primer pairs asdescribed in Example 5. The mutant PS-1 allele with theneomycin-selectable marker excised was designated PS-1^(P264L).Successful excision occurred in one founder mouse that generatedheterozygous (PS-1^(P264L/+)) and homozygous (PS-1^(P264L/P264L)) linesof mice. PS-1^(P264L/+) mice were crossed with Tg2576 mice andAPP^(NLh/NLh) mice to fiuther study the effects of the P264L mutation onAβ production and deposition.

Example 7 Northern And Western Blots

[0132] PS-1^(+/+), PS-1^(nP264L/nP264L), and PS-1^(P264L/P264L) mice,aged 2-6 months were used for evaluating mRNA and protein levels ofPS-1. Total RNA was extracted from one-half brain by homogenization inRNAzol B (Tel-Test, Friendswood, Tex.). Messenger RNA was selected withOligotex columns (Qiagen, Valencia, Calif.). Equal volumes of mRNA weremixed with loading buffer (NortherMAX-Gly, Ambion, Austin, Tex.) heatedto 50° C. for 30 min, separated on a 0.7% agarose gel, and transferredto a nylon membrane. PS-1 mRNA was detected with a ³²P-dUTP-labeledriboprobe representing the 3′ end of human PS-1: nucleotides 1083-1428cloned into a pGEM-T vector (Promega, Madison, Wis.). The same blot washybridized with a GAPDH probe (Ambion) for normalization. To visualizemRNAs, the membrane was exposed to a phosphor screen, scanned on a Storm840 PhosphorImager, and densitometry performed with ImageQuaNT software(Molecular Dynamics, Sunnyvale, Calif.).

[0133] One-half brain was homogenized in 2.5 ml of buffer containing 10mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% SDS, 0.25% deoxycholate,0.25% NP-40, and protease inhibitors (5 mM PMSF, 10 μg/ml aprotinin, 10μg/ml leupeptin, 10 μg/ml pepstatin) (Lee et al., Nature Med., 1997, 3,756-760). Protein concentration was determined by BCA assay (Pierce,Rockford, Ill.). Total brain lysates were mixed with reducing loadingbuffer and heated at 37° C. for 45 min. Fifty μg total protein of eachsample was separated by electrophoresis on NuPAGE 10% polyacrylamidegels (Novex, San Diego, Calif.), and transferred to nitrocellulose.Membranes were blocked overnight at 4° C. in Tris buffered saline (TBS)with 0.05% Tween-20 and 5% nonfat dry milk. PS-1 was detected withrabbit polyclonal antibodies diluted in the same solution. TheC-terminal fragment was detected with antibody B17.2 (De Strooper etal., J. Biol. Chem., 1997, 272, 3590-3598) at 1:2000 dilution. B17.2 wasraised against amino acid residues 300-315 (EGDPEAQRRVSKNSKY; SEQ IDNO:17) in the hydrophilic loop domain of human PS-1. The N-terminalfragment was detected with CP160 at 1:500. CP160 was generated using a6X-histidine tagged N-terminal fragment of human PS-1 (amino acids1-80), expressed in bacteria with a pQE-9 plasmid (Qiagen). Thesynthetic PS-1 N-terminal fragment was purified using Ni-NTA agarose(Qiagen) and SDS-PAGE. Peptide was cut out of acrylamide gels forinjection into rabbits. The IgG fraction of CP160 was affinity purifiedand used for blotting. The primary antibodies were detected withhorseradish peroxidase-conjugated anti-rabbit secondary antibodies (NewEngland Biolabs, Beverly, Mass.). Blots were reacted withchemiluminescent reagent (LumiGLO, New England Biolabs) and exposed toHyperfilm (Amersham, Arlington Heights, Ill.). Films were scanned anddensitometry performed with RFLP2.1 software (Scanalytics, Fairfax,Va.).

[0134] Northern blot analysis demonstrated a 3.1 kb band for PS-1 mRNA.PS-1 mRNA levels were normalized for loading differences with GAPDH mRNAlevels. The presence of the PGK-neo gene in the PS-1^(nP264L/nP264L)mice resulted in levels of PS-1 mRNA that were 20% of wild type levels(data not shown). mRNA levels in PS-1^(P264L/P264L) mice were 100% ofPS-1^(+/+) mice (data not shown). Thus, removal of the PGK-neo cassettereturned PS-1 mRNA levels to normal levels.

[0135] Western blotting demonstrated an N-terminal PS-1 fragment of ˜30kDa using antibody CP160 and a C-terminal PS-1 fragment of ˜20 kDa usingantibody B17.2 in all three genotypes. Blotting with CP160, preabsorbedwith antigen, eliminated the ˜30 kDa N-terminal band (data not shown).Specificity of B17.2 for the C-terminal PS-1 fragment has beenpreviously demonstrated (De Strooper et al., J. Biol. Chem., 1997, 272,3590-3598). Because PS-1 mRNA levels were reduced in thePS-1^(nP264L/nP264L) mice, the level of PS-1 protein was also reduced toapproximately 15-20% of normal levels (data not shown). In spite ofnormal mRNA expression of mutant PS-1 in the PS-1^(P264L/P264L) mice,PS-1 protein levels were reduced by about 50% (data not shown). Both theN- and C-terminal fragments were reduced to a similar degree. These dataindicate that the PS-1^(P264L) mutation affects PS-1 protein levelseither via effects on translation, processing of the full length PS-1protein, or stability of the cleaved fragments. Reports have describedvariable reductions in the N-terminal fragment and/or accumulation ofholoprotein due to some FAD mutations in PS-1 (Mercken et al., FEBSLett., 1996, 389, 297-303; Murayama et al., Neurosci. Lett., 1997, 229,61-64; Levey et al., Ann. Neurol., 1997, 41, 742-753; Murayama et al.,Prog. Neuro-Psychopharmacol. Biol. Psychiatr., 1999, 23, 905-913;Takahashi et al., Neurosci. Lett., 1999, 260, 121-124). In contrast, avariety of PS-1 FAD mutations were found to cause no reductions infragment formation in FAD patients, in transfected cells, and intransgenic or gene-targeted mice (Hendriks et al., NeuroReport, 1997, 8,1717-1721; Lee, et al., Nature Med., 1997, 3, 756-760; Podlisny et al.,Neurobiol. Dis., 1997, 3, 325-337; Guo et al., Nature Med., 1999, 5,101-106; Lévesque et al., Molec. Med., 1999, 5, 542-554; Vanderhoeven etal., Neurosci. Lett., 1999, 274, 183-186; Borchelt et al., Neuron, 1996,17, 1005-1013; Duff et al., Nature, 1996, 383, 710-713; Citron et al.,Nature Med., 1997, 3, 67-72; Nakano et al., Eur. J. Neurosci., 1999, 11,2577-2581). The level of reduction in the N-terminal fragment for theP264L mutation was to about 35-40% of wild type PS-1 values intransfected PC12 cells (Murayama et al., Prog. Neuro-Psychopharmacol.Biol. Psychiatr., 1999, 23, 905-913), consistent with the degree ofreduction that has been seen in the PS-1^(P264L/P264L) mice comparedwith PS-1^(+/+) mice.

Example 8 Aβ40- and 42-specific ELISAs

[0136] Half brains from predepositing double gene-targeted mice(APP^(NLh/NLh) mice at 1-6 months, APP^(NLh/NLh)×PS-1^(P264L/+) mice at5 months, and APP^(NLh/NLh)×PS-1^(P264L/P264L) mice at 1-2 months) andpredepositing Tg2576 mice (Hsiao et al., Science, 1996, 274, 99-102) at2-4 months were frozen on dry ice and stored at −70° C. Additional halfbrains from predepositing Tg2576 mice crossed with PS-1^(P264L/+) mice(Tg2576×PS-1^(+/+) mice at 2-4 months, Tg2576×PS-1^(P264L/+) mice at 2months, and Tg2576×PS-1^(P264L/P264L) mice at 1 month) were similarlyprepared. Half brains were homogenized in 4 ml of 0.2% diethylamine and50 mM NaCl and centrifuged at 100,000× g. The supernatants wereneutralized to pH 8 with 2 M Tris-HCl, assayed for protein concentrationby the BCA method (Pierce, Rockford, Ill.), and diluted 1:1 in 5% fetalclone serun (HyClone, Logan, Utah) and 1% nonfat dry milk in TBS. TheAβ42-specific ELISA was runas previously described (Savage et al., J.Neurosci., 1998, 18, 1743-1752). The Aβ40-specific ELISA was modified(Savage et al., J. Neurosci., 1998, 18, 1743-1752) so that the captureantibody was 6E10 (Senetek, Napa, Calif.) and the detecting antibody wasselective for Aβ40 (BioSource International, Camarillo, Calif.). ELISAsignals were reported as nanograms of Aβ per milligram of totalextracted protein based upon standard curves generated using Aβ40 or 42(Bachem, King of Prussia, Pa.).

[0137] Table 2 shows the effect of the PS-1^(P264L) mutation on Aβ40 andAβ42 levels in the brains of APP^(NLh/NLh) mice before the appearance ofAβ deposition. The PS-1^(P264L) mutation did not have a significanteffect on the level of Aβ40. One copy of the PS-1^(P264L) mutationslightly elevated Aβ42 but the effect of the mutation was significantonly in the APP^(NLh/NLh)×PS-1^(P264L/P264L) mice compared with theAPP^(NLh/NLh)×PS-1^(+/+) mice. This increase in Aβ42 levels caused asignificant elevation in the ratio of Aβ42 to Aβ40 in theAPP^(NLh/NLh)×PS-1^(P264L/P264L) mice relative toAPP^(NLh/NLh)×PS-1^(+/+) and APP^(NLh/NLh)×PS-1^(P264L/+) mice. Tg2576mice had markedly more Aβ40 and Aβ42 than theAPP^(NLh/NLh)×PS-1^(P264L/P264L) mice but the ratio of Aβ42/40 wassimilar in the Tg2576 and APP^(NLh/NLh)×PS-1^(+/+) mice.

[0138] Table 3 shows the effect of the PS-1^(P264L) mutation on Aβ40 andA,42 levels in the brains of Tg2576 mice before the appearance of Aβdeposition. The PS-1^(P264L) mutation did not have a significant effecton the level of Aβ40. One copy of the PS-1^(P264L) mutation slightlyelevated Aβ42 but the effect of the mutation was significant only in theTg2576×PS-1^(P264L/P264L) mice compared with the Tg2576×PS-1^(+/+) mice.The increase in Aβ42 levels caused a significant elevation in the ratioof Aβ42 to Aβ40 in the Tg2576×PS-1^(P264L/P264L) mice relative toTg2576×PS-1^(+/+) mice. Thus, the effect of the PS-1^(P264L) mutation onAβ levels was similar for the Tg2576 and APP^(NLh/NLh) mice. TABLE 2Aβ40 and 42 Levels in Predepositing APP^(NLh/NLh) with PS-1^(P264L)Mutations and Tg2576 Mice Aβ40 Aβ42 PS-1 Genotype Age (days) N (ng/mgprotein) (ng/mg protein) Aβ42/40 Ratio APP^(NLh/NLh) Mice withPS-1^(P264L) Mutations X PS-1^(+/+) 103 8 0.40 ± 0.03 0.08 ± 0.01 0.18 ±0.03 X PS-1^(P264L/+) 138 5 0.47 ± 0.08 0.11 ± 0.03 0.22 ± 0.02 XPS-1^(P264L/P264L) 54 10 0.40 ± 0.03  0.15 ± 0.01*   0.37 ± 0.01**Tg2576 Mice on C57B6/SJL Background 99 6 1.52 ± 0.17 0.29 ± 0.05 0.18 ±0.01

[0139] TABLE 3 Aβ40 and 42 Levels in Predepositing Tg2576 Mice withPS-1^(P264L) Mutations Tg2576 Mice with PS-1^(P264L) Mutations Aβ40 Aβ42PS-1 Genotype Age (days) N (ng/mg protein) (ng/mg protein) Aβ42/40 RatioX PS-1^(+/+) 92 6 1.73 ± 0.10 0.27 ± 0.2  0.16 ± 0.01 X PS-1^(P264L/+)62 5 2.15 ± 0.19 0.44 ± 0.07 0.20 ± 0.02 X PS-1^(P264L/P264L) 31 8 1.72± 0.22  0.57 ± 0.08*   0.33 ± 0.01**

Example 9 Immunohistochemistry and Histology

[0140] PS-1^(P264L/P264L) mice were examined at 12 months of age.APP^(NLh/NLh) mice that were PS-1^(+/+), PS-1^(P264L/+), orPS-1^(P264L/P264L), aged 3, 6, 9, 12, 15, and 18 months of age wereevaluated. Additional mice examined were Tg2576 and were PS-1^(+/+),PS-1^(P264L/+), or PS-1^(P264L/P264L), aged 1, 2, 4, 6, 9, 12, 15, and18 months of age. Other Tg2576 mice maintained by crossing to C57B6/SJLmice were also examined at 6, 9, 12, 15, 18, and 21 months of age. Micewere perfused with Ringer's solution and the brains removed andhemisected. One-half of each brain was immersed in 70% ethanol and 150mM NaCl for 48 hours, paraffin embedded and sectioned in the sagittalplane at 10 μm. Sets of 16 sections taken at intervals of 200 μm werestained to demonstrate Aβ deposits by immunohistochemistry. Antibodiesused were 1153, a rabbit polyclonal antibody generated against aminoacids 1-28 of human Aβ (Savage et al., Neuroscience, 1994, 60, 607-619)and monoclonal antibodies 4G8 and 6E10 (Senetek). Sections werepretreated with 80% formic acid for 4G8, not pretreated for 1153 and6E10, and were reacted with the primary antibodies overnight at 1:1,000.Antibodies were complexed using biotinylated secondary antibodies(1:100), linked using streptavidin labeled horseradish peroxidase(BioGenex, San Ramon, Calif.), and visualized using nickel-intensified3,3′-diaminobenzidene. Non-transgenic mice, as well as pre-absorbedprimary antisera, served as staining controls. Additional sets ofsections were stained using thioflavine S and examined with afluorescence microscope.

[0141] Plaque load was quantified in neocortex in one set of 16 sectionsstained with antibody 1153 using the CastGrid system (Olympus,Copenhagen, Denmark). Volume of neocortex and percent volume ofneocortex occupied by Aβ deposits were determined stereologically bypoint counting (Weibel et al., (1979) Stereological methods, vol. 1:practical methods for biological morphometry, 415 pp. London: AcademicPress). Representative results are shown in Table 4. TABLE 4 Aβ PlaqueLoad (% Volume Fraction) in Neocortex at 6 Months of Age Genotype %Plaque Load Tg2576 (C57B6/SJL) 0.0018 Tg2576 × PS-1^(+/+) 0.0016 Tg2576× PS-1^(P264L/+) 2.92 Tg2576 × PS-1^(P264L/P264L) 9.09 APP^(NLh/NLh) ×PS-1^(+/+) 0 APP^(NLh/NLh) × PS-1^(P264L/+) 0 APP^(NLh/NLh) ×PS-1^(P264L/P264L) 0.026

[0142] Extracellular Aβ deposition was markedly accelerated in Tg2576mice that were PS-1^(P264L/+) or PS-1^(P264L/P264L) compared with thosethat were PS-1^(+/+). In Tg2576×PS-1^(P264L/+) mice, Aβ deposition wasnot noted at 2 months of age but was present at 4 months of age. InTg2576×PS-1^(P264L/P264L) mice, Aβ deposition was not present at 1 monthof age but was present at 2 months of age. Tg2576×PS-1^(+/+) mice didnot show Aβ deposition until 6 months of age, and the amount wascomparable to that seen in 6-month-old Tg2576 mice maintained on theC57B6/SJL background. Aβ plaque load visualized with antibody 1153increased dramatically in neocortex of the Tg2576×PS-1^(P264L/+) andTg2576×PS-1^(P264L/P264L) mice at later ages (data not shown).Tg2576×PS-1^(P264L/+) mice at 4 months and Tg2576×PS-1^(P264L/P264L)mice at 2 months had numerous deposits that stained with 4G8 orthioflavine S, indicating that the earliest deposits contained compact,fibrillar amyloid.

[0143] In addition to the acceleration in deposition seen inTg2576×PS-1^(P264L/P264L) mice, another difference was the regionaldistribution of deposition. Comparing 6-month-oldTg2576×PS-1^(P264L/P264L) mice, 9-month-old Tg2576×PS-1^(P264L/+) mice,and 18-month-old Tg2576 (C57B6/SJL background) mice, the density ofdeposition was similar in telencephalic structures (data not shown).However, in subcortical structures of the Tg2576×PS-1^(P264L/P264L) micethe amount of Aβ deposition was much greater than in theTg2576×PS-1^(P264L/+) and Tg2576 mice (data not shown).

[0144] Aβ deposition in the APP^(NLh/NLh) mice has been assessed out to22 months of age. No evidence for deposition was found. Similarly, nodeposition was found in PS-1^(P264L/P264L) mice that were wild type formouse APP at 12 months of age. Extremely rare Aβ deposition was noted inthe cortex of APP^(NLh/NLh)×PS-1^(P264L/+) mice using both antibody 1153and thioflavine S at 12 months of age. At 18 months of age Aβ depositsin these mice were more numerous and larger.

[0145] Two copies of the PS-1^(P264L) mutation resulted in an increasein Aβ42 in the APP^(NLh/NLh)×PS-1^(P264L/P264L) mouse (Table 2) and haveresulted in Aβ deposition at an early age. Aβ deposition was not foundat 3 months of age but was present at 6 months inAPP^(NLh/NLh)×PS-1^(P264L/P264L) mice. Deposition increased with age inthe APP^(NLh/NLh)×PS-1^(P264L/P264L) mice.

[0146] The disclosures of each patent, patent application andpublication cited or described in this document are hereby incorporatedherein by reference, in their entirety.

[0147] Various modifications of the invention, in addition to thosedescribed herein, will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

1 17 1 21 DNA Artificial Sequence Oligonucleotide Primer 1 ctcatcttggctgtgatttc a 21 2 18 DNA Artificial Sequence Oligonucleotide Primer 2gttgtgttcc agtctcca 18 3 19 DNA Artificial Sequence OligonucleotidePrimer 3 atttagtggc tgttttgtg 19 4 20 DNA Artificial SequenceOligonucleotide Primer 4 aggagtaaat gagagctgga 20 5 21 DNA ArtificialSequence Oligonucleotide Primer 5 tgaaatcaca gccaagatga g 21 6 22 DNAArtificial Sequence Oligonucleotide Primer 6 gcactcctga tctggaattt tg 227 48 DNA Artificial Sequence Oligonucleotide Primer 7 ggaaagaatgcggccgctgt cgacgttaac atgcatataa cttcgtat 48 8 47 DNA ArtificialSequence Oligonucleotide Primer 8 gctctcgaga taacttcgta tagcatacattatacgaagt tatatgc 47 9 33 DNA Artificial Sequence OligonucleotidePrimer 9 cgttctagaa taacttcgta taatgtatgc tat 33 10 33 DNA ArtificialSequence Oligonucleotide Primer 10 cgtggatcca taacttcgta tagcatacat tat33 11 27 DNA Artificial Sequence Oligonucleotide Primer 11 ttgtgtcttaagggtccgct tcgtatg 27 12 27 DNA Artificial Sequence OligonucleotidePrimer 12 cggaccctta agacacaaaa cagccac 27 13 20 DNA Artificial SequenceOligonucleotide Primer 13 ggattgcacg caggttctcc 20 14 20 DNA ArtificialSequence Oligonucleotide Primer 14 ccggcttcca tccgagtacg 20 15 24 DNAArtificial Sequence Oligonucleotide Primer 15 cccgtggagg aggtcagaag tcag24 16 21 DNA Artificial Sequence Oligonucleotide Primer 16 ttacgggttgagccatgaat g 21 17 16 PRT Homo sapiens 17 Glu Gly Asp Pro Glu Ala GlnArg Arg Val Ser Lys Asn Ser Lys Tyr 1 5 10 15

What is claimed is:
 1. A gene-targeted, non-human mammal heterozygousfor a human Familial Alzheimer's Disease (FAD) mutation comprising ahuman mutation of the presenilin-1 (PS-1 gene), a human FAD Swedishmutation, and a humanized Aβ gene.
 2. A gene-targeted, non-human mammalhomozygous for a human Familial Alzheimer's Disease (FAD) mutationcomprising a human mutation of the presenilin-1 (PS-1 gene), a human FADSwedish mutation, and a humanized Aβ gene.
 3. The mammal of claim 1wherein said mutation of said PS-1 gene is P264L.
 4. The mammal of claim2 wherein said mutation of said PS-1 gene is P264L.
 5. The mammal ofclaim 1 wherein said mammal is a rodent.
 6. The mammal of claim 5wherein said mammal is a mouse.
 7. The mammal of claim 2 wherein saidmammal is a rodent.
 8. The mammal of claim 7 wherein said mammal is amouse.
 9. Generational offspring of the mammal of claim 1 wherein saidmutant PS-1 gene is expressed.
 10. Generational offspring of the mammalof claim 2 wherein said mutant PS-1 gene is expressed.
 11. A method forscreening chemical compounds for the ability to decrease in vivo levelsof Aβ peptide, said method comprising the steps of: a) administeringsaid chemical compound to the mammal of claim 1; and b) measuring theamount of Aβ peptide in a tissue sample from said mammal, wherein adecrease in the amount of Aβ peptide in said tissue sample is indicativeof a chemical compound that has the ability to decrease in vivo levelsof said Aβ peptide.
 12. A method for screening chemical compounds forthe ability to decrease in vivo levels of the Aβ peptide, said methodcomprising the steps of: a) administering said chemical compound to themammal of claim 2; and b) measuring the amount of Aβ peptide in a tissuesample from said mammal, wherein a decrease in the amount of Aβ peptidein said tissue sample is indicative of a chemical compound that has theability to decrease in vivo levels of said Aβ peptide.
 13. A method forscreening chemical compounds for the ability to decrease in vivo levelsof the Aβ peptide, said method comprising the steps of: a) administeringsaid chemical compound to the mammal of claim 9; and b) measuring theamount of Aβ peptide in a tissue sample from said mammal, wherein adecrease in the amount of Aβ peptide in said tissue sample is indicativeof a chemical compound that has the ability to decrease in vivo levelsof said Aβ peptide.
 14. A method for screening chemical compounds forthe ability to decrease in vivo levels of the Aβ peptide, said methodcomprising the steps of: a) administering said chemical compound to themammal of claim 10; and b) measuring the amount of Aβ peptide in atissue sample from said mammal, wherein a decrease in the amount of Aβpeptide in said tissue sample is indicative of a chemical compound thathas the ability to decrease in vivo levels of said Aβ peptide.
 15. Themethod of claim 11 wherein said tissue sample is selected from the groupconsisting of brain tissue, non-brain tissue and body fluids.
 16. Themethod of claim 12 wherein said tissue sample is selected from the groupconsisting of brain tissue, non-brain tissue and body fluids.
 17. Themethod of claim 13 wherein said tissue sample is selected from the groupconsisting of brain tissue, non-brain tissue and body fluids.
 18. Themethod of claim 14 wherein said tissue sample is selected from the groupconsisting of brain tissue, non-brain tissue and body fluids.
 19. Amethod for identifying a compound for treating Alzheimer's diseasecomprising the steps of: a) administering a compound to the mammal ofclaim 1; and b) measuring the amount of Aβ peptide in a tissue samplefrom said mammal, wherein a decrease in the amount of Aβ peptide in saidtissue sample is indicative of a compound that can be used to treatAlzheimer's disease.
 20. A method for identifiing a compound fortreating Alzheimer's disease comprising the steps of: a) administering acompound to the mammal of claim 2; and b) measuring the amount of Aβpeptide in a tissue sample from said mammal, wherein a decrease in theamount of Aβ peptide in said tissue sample is indicative of a compoundthat can be used to treat Alzheimer's disease.
 21. A method foridentifying a compound for treating Alzheimer's disease comprising thesteps of: a) administering a compound to the mammal of claim 9; and b)measuring the amount of Aβ peptide in a tissue sample from said mammal,wherein a decrease in the amount of Aβ peptide in said tissue sample isindicative of a compound that can be used to treat Alzheimer's disease.22. A method for identifying a compound for treating Alzheimer's diseasecomprising the steps of: a) administering a compound to the mammal ofclaim 10; and b) measuring the amount of Aβ peptide in a tissue samplefrom said mammal, wherein a decrease in the amount of Aβ peptide in saidtissue sample is indicative of a compound that can be used to treatAlzheimer's disease.
 23. The method of claim 19 wherein said tissuesample is selected from the group consisting of brain tissue, non-braintissue and body fluids.
 24. The method of claim 20 wherein said tissuesample is selected from the group consisting of brain tissue, non-braintissue and body fluids.
 25. The method of claim 21 wherein said tissuesample is selected from the group consisting of brain tissue, non-braintissue and body fluids.
 26. The method of claim 22 wherein said tissuesample is selected from the group consisting of brain tissue, non-braintissue and body fluids.
 27. A method of treating an individual suspectedof having Alzheimer's disease comprising administering to saidindividual an effective Alzheimer's disease treatment amount of acompound identified by the method of claim
 19. 28. A method of treatingan individual suspected of having Alzheimer's disease comprisingadministering to said individual an effective Alzheimer's diseasetreatment amount of a compound identified by the method of claim
 20. 29.A method of treating an individual suspected of having Alzheimer'sdisease comprising administering to said individual an effectiveAlzheimer's disease treatment amount of a compound identified by themethod of claim
 21. 30. A method of treating an individual suspected ofhaving Alzheimer's disease comprising administering to said individualan effective Alzheimer's disease treatment amount of a compoundidentified by the method of claim
 22. 31. A compound identified by themethod of claim
 11. 32. A compound identified by the method of claim 12.33. A compound identified by the method of claim
 13. 34. A compoundidentified by the method of claim
 14. 35. A compound identified by themethod of claim
 19. 36. A compound identified by the method of claim 20.37. A compound identified by the method of claim
 21. 38. A compoundidentified by the method of claim
 22. 39. A gene-targeted, non-humanmammal heterozygous for a human Familial Alzheimer's Disease (FAD)mutation comprising a human mutation of the presenilin-1 (PS-1 gene),and a human transgenic for Swedish APP695.
 40. A gene-targeted,non-human mammal homozygous for a human Familial Alzheimer's Disease(FAD) mutation comprising a human mutation of the presenilin-1 (PS-1gene), and a human transgenic for Swedish APP695.
 41. The mammal ofclaim 39 wherein said mutation of said PS-1 gene is P264L.
 42. Themammal of claim 40 wherein said mutation of said PS-1 gene is P264L 43.The mammal of claim 39 wherein said mammal is a rodent.
 44. The mammalof claim 43 wherein said mammal is a mouse.
 45. The mammal of claim 40wherein said mammal is a rodent.
 46. The mammal of claim 45 wherein saidmammal is a mouse.
 47. Generational offspring of the mammal of claim 39wherein said mutant PS-1 gene is expressed.
 48. Generational offspringof the mammal of claim 40 wherein said mutant PS-1 gene is expressed.49. A method for screening chemical compounds for the ability todecrease in vivo levels of the Aβ peptide, said method comprising thesteps of: a) administering said chemical compound to the mammal of claim39; and b) measuring the amount of Aβ peptide in a tissue sample fromsaid mammal, wherein a decrease in the amount of Aβ peptide in saidtissue sample is indicative of a chemical compound that has the abilityto decrease in vivo levels of said Aβ peptide.
 50. A method forscreening chemical compounds for the ability to decrease in vivo levelsof the Aβ peptide, said method comprising the steps of: a) administeringsaid chemical compound to the mammal of claim 40; and b) measuring theamount of Aβ peptide in a tissue sample from said mammal, wherein adecrease in the amount of Aβ peptide in said tissue sample is indicativeof a chemical compound that has the ability to decrease in vivo levelsof said Aβ peptide.
 51. A method for screening chemical compounds forthe ability to decrease in vivo levels of the Aβ peptide, said methodcomprising the steps of: a) administering said chemical compound to themammal of claim 47; and b) measuring the amount of Aβ peptide in atissue sample from said mammal, wherein a decrease in the amount of Aβpeptide in said tissue sample is indicative of a chemical compound thathas the ability to decrease in vivo levels of said Aβ peptide.
 52. Amethod for screening chemical compounds for the ability to decrease invivo levels of the Aβ peptide, said method comprising the steps of: a)administering said chemical compound to the mammal of claim 48; and b)measuring the amount of Aβ peptide in a tissue sample from said mammal,wherein a decrease in the amount of Aβ peptide in said tissue sample isindicative of a chemical compound that has the ability to decrease invivo levels of said Aβ peptide.
 53. The method of claim 49 wherein saidtissue sample is selected from the group consisting of brain tissue,non-brain tissue and body fluids.
 54. The method of claim 50 whereinsaid tissue sample is selected from the group consisting of braintissue, non-brain tissue and body fluids.
 55. The method of claim 51wherein said tissue sample is selected from the group consisting ofbrain tissue, non-brain tissue and body fluids.
 56. The method of claim52 wherein said tissue sample is selected from the group consisting ofbrain tissue, non-brain tissue and body fluids.
 57. A method foridentifying a compound for treating Alzheimer's disease comprising thesteps of: a) administering a compound to the mammal of claim 39; and b)measuring the amount of Aβ peptide in a tissue sample from said mammal,wherein a decrease in the amount of Aβ peptide in said tissue sample isindicative of a compound that can be used to treat Alzheimer's disease.58. A method for identifying a compound for treating Alzheimer's diseasecomprising the steps of: a) administering a compound to the mammal ofclaim 40; and b) measuring the amount of Aβ peptide in a tissue samplefrom said mammal, wherein a decrease in the amount of Aβ peptide in saidtissue sample is indicative of a compound that can be used to treatAlzheimer's disease.
 59. A method for identifying a compound fortreating Atzheimer's disease comprising the steps of: a) administering acompound to the mammal of claim 47; and b) measuring the amount of Aβpeptide in a tissue sample from said mammal, wherein a decrease in theamount of Aβ peptide in said tissue sample is indicative of a compoundthat can be used to treat Alzheimer's disease.
 60. A method foridentifying a compound for treating Alzheimer's disease comprising thesteps of: a) administering a compound to the mammal of claim 48; and b)measuring the amount of Aβ peptide in a tissue sample from said mammal,wherein a decrease in the amount of Aβ peptide in said tissue sample isindicative of a compound that can be used to treat Alzheimer's disease.61. The method of claim 57 wherein said tissue sample is selected fromthe group consisting of brain tissue, non-brain tissue and body fluids.62. The method of claim 58 wherein said tissue sample is selected fromthe group consisting of brain tissue, non-brain tissue and body fluids.63. The method of claim 59 wherein said tissue sample is selected fromthe group consisting of brain tissue, non-brain tissue and body fluids.64. The method of claim 60 wherein said tissue sample is selected fromthe group consisting of brain tissue, non-brain tissue and body fluids.65. A method of treating an individual suspected of having Alzheimer'sdisease comprising administering to said individual an effectiveAlzheimer's disease treatment amount of a compound identified by themethod of claim
 57. 66. A method of treating an individual suspected ofhaving Alzheimer's disease comprising administering to said individualan effective Alzheimer's disease treatment amount of a compoundidentified by the method of claim
 58. 67. A method of treating anindividual suspected of having Alzheimer's disease comprisingadministering to said individual an effective Alzheimer's diseasetreatment amount of a compound identified by the method of claim
 59. 68.A method of treating an individual suspected of having Alzheimer'sdisease comprising administering to said individual an effectiveAlzheimer's disease treatment amount of a compound identified by themethod of claim
 60. 69. A compound identified by the method of claim 49.70. A compound identified by the method of claim
 50. 71. A compoundidentified by the method of claim
 51. 72. A compound identified by themethod of claim
 52. 73. A compound identified by the method of claim 57.74. A compound identified by the method of claim
 58. 75. A compoundidentified by the method of claim
 59. 76. A compound identified by themethod of claim 60.